Mutant human and simian immunodeficiency virus ENV proteins with reduced immunosuppressive properties

ABSTRACT

A pharmaceutical composition includes, as active substance a mutated lentiviral ENV protein, substantially devoid of immunosuppressive properties or a variant of the mutated lentiviral ENV protein or a fragment of the above proteins, in association with a pharmaceutically acceptable carrier.

FIELD OF THE INVENTION

The present invention relates to mutated lentiviral ENV proteins and their use as drugs.

BACKGROUND OF THE INVENTION

Lentivirus is a genus of slow viruses of the Retroviridae family, characterized by a long incubation period. Lentiviruses can deliver a significant amount of genetic information into the DNA of the host cell and have the unique ability among retroviruses of being able to replicate in non-dividing cells, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.

Human immunodeficiency virus (HIV) is according to WHO one of the most serious health crisis the world faces today. AIDS has killed more than 25 million people since 1981. In the most severely affected countries, the average life expectancy is now declining to 49 years of age—13 years less than in the absence of AIDS. According to UNAIDS an estimated number of 39.5 million people were living with HIV virus in 2006 and 4.3 million were infected in 2006. In many regions new infections are heavily concentrated in the younger generations (15-24 years of age). Access to treatment and care has greatly increased in recent years. Determining real time trends to HIV incidence and in particular the impact of prevention programmes ideally requires long studies of a large number of people. Given the practical difficulties of conducting such studies focus has been placed on young women and their infants. Children living with HIV typically acquire infection through a mother-to-child-transmission (MTCT), which occur during pregnancy, delivery or during breastfeeding. Renewed efforts are urgently required to increase access to comprehensive and integrated programs to prevent HIV infection in infants and young children, which will indicate a route to HIV-free generations.

There are two known types of HIV; HIV-1 and HIV-2 that infect humans. They belong to a group of retroviruses called Lentiviruses and a virus similar to HIV has been found in African monkeys. Retroviruses transfer their genes from a producer cell to a target cell as a genomic RNA transcript, which is reverse-transcribed after infection and integrated into the DNA genome of the target cell.

The first person with a documented HIV-infection died in 1959. In the early 1980s doctors in the US become aware, that more and more patients suffered from abnormal infections and showed signs of immune failure. The syndrome was named Acquired Immune Deficiency Syndrome (AIDS) and it was soon after discovered that HIV was the causative agent for the observed destruction of the immune system. Initially patients were offered a treatment based solely on pain relief and almost all inevitably died. In mid 1990s there were two important breakthroughs in treatment. Firstly, a new group of antiretroviral agents were discovered and secondly it became possible to measure the amount of HIV virus in blood. These two advances made it possible to treat patients with a combination of different agents and doctors were able to check, whether the treatment actually worked. The result was that the immune system of infected patients gradually became normal and patients lived longer. Today infected people in Western countries are having the same level of quality of life as those not infected and they are able to have children, although the economical and psychological consequences of having HIV are huge. The situation is, however, even more severe in developing countries, where more than 95% of those people infected with HIV/AIDS are living. Worldwide more than 25 million people have died from AIDS in the last 25 years.

Approximately 95% of the people who get infected today live in the developing countries, where expensive antiviral drugs are not available. Therefore, there is an urgent need for an effective vaccine—the only effective solution to the uncontrolled HIV pandemic. During the last few years research has brought up new knowledge on the fundamental biology of HIV-virus which is leading to new antiviral drugs and strategies for vaccine design. In spite of these substantial advances, an effective vaccine does not yet exist. Only attenuated (that is live but weakened) HIV-strains has been able to provide immunity in primate studies even though they will never reach a required safety profile suitable for mass vaccination.

The replication process for HIV-1 has an error rate of about one per 10,000 base pairs. Since the entire viral genome is just under 10,000 base pairs, it is estimated that on average one error is introduced into the HIV-1 genome at each viral replication cycle.

This high mutation rate contributes to extensive variability of the viruses inside any one person and an even wider variability across populations.

This variability has resulted in three HIV-1 variants being described, and the subspecies of virus called “clades.” The distinctions are based on the structure of the envelope proteins, which are especially variable. The M (for major) variant is by far the most prevalent world wide. Within the M variant are clades A, B. C, D, E, F. G H. I, J and K, with clades A through E representing the vast majority of infections globally. Clades A, C and D are dominant in Africa, while clade B is the most prevalent in Europe, North and South America and Southeast Asia. Clades E and C are dominant in Asia. These clades differ by as much as 35%. Another variant is clade O, which is observed in Cameroun isolates of HIV-1. The greatest variation in structure is seen in the envelope proteins gp120 and gp41. There are two important results from the very high mutation rate of HIV-1 that have profound consequences for the epidemic. First, the high mutation rate is one of the mechanisms that allow the virus to escape from control by drug therapies. These new viruses represent resistant strains. The high mutation rate also allows the virus to escape the patient's immune system by altering the structures that are recognized by immune components. An added consequence of this extensive variability is that the virus can also escape from control by vaccines, and therefore makes it difficult to find vaccines based on envelope proteins which are effective.

Moreover, the virus produces proteins having immunosuppressive properties, allowing to escape the patient's immune system survey. Thus, cell expressing such proteins become “invisible” to the immune system.

Consequently for a vaccine, there is a need to provide proteins as antigens having lost, or substantially lost, their immunosuppressive functions, in order to generate an efficient response. This will enable the individuals once infected by the virus to allow the immune system to destroy the infected cells and prevent/cure the infection.

Prior art has already intended to provide such proteins.

For instance, the international application WO 2005/095,442 (Inventors: Renard, Mangeney & Heidmann) discloses mutations in the immunosuppressive domains of endogenous retroviruses (ERV) or onco retroviruses, such as HTLV or FeLV, ENV proteins. This document demonstrates that mutations at a specific position abolish the immunosuppressive properties of ENV proteins of ERV or onco retroviruses. However, the international application WO 2005/095, 442 never mentions or suggests that the mutations made in the immunosuppressive domain of ERV- or onco retroviruses ENV proteins can be transposable to lentiviral ENV proteins.

The international application WO 2010/022,740 discloses an extremely wide consensus sequence of a region of HIV ENV protein, described as follows:

-   -   X(1-22)-C(23)-X(24-28)-C(29)-(X30-50)         wherein the amino acid residues of the consensus sequence are         selected from the groups of residues consisting of:         X(1): L, S, R, P, F, A, V, M, and I; and         X(2): Q, R, K, H, L, M, and P; and         X(3): A, T, V, H, S, R, Q, G, M, and E; and         X(4): R, K, G, E, T, S, C, M, and H; and         X(5): V, I, L, D, A, S, F, M, and G; and         X(6): L, Q, V, M, P, W, T, and I; and         X(7): A, S, T, V, L, G, F, D, M, and E; and         X(8): V, L, I, M, A, W, K, G, and E; and         X(9): E, K, G, D, A, V, M, and F; and         X(10): X; and         X(11): Y, L, F, H, C, I, T, M, and N; and         X(12): L, I, V, M, Q, P, T, Y, and A; and         X(13): K, R, Q, G, S, E, H, W, T, V, M, N, Z, Y, A, P, and C;         and         X(14): D, N, G, E, Y, V, S, H, A, M, and I; and         X(15): Q, R, H, K, P, L, M, and N; and         X(16): Q, K, R, T, H, E, S, P, M, and L; and         X(17): L, F, I, R, V, P, S, M, and H, and         X(18): L, M, P, I, H, and S; and         X(19): X; and         X(20): I, L, M, V, S, F, T, D, A, R, P, and J; and         X(21): W, R, G, F, L, M, and T; and         X(22): G, D, A, R, M, and C; and         X(24): X; and         X(25): G, R, E, N, A, M, and D; and         X(26): K, R, N, E, Q, T, S, I, M, and G; and         X(27): L, H, I, T, V, F, R, Q, S, P, A, J, M, and Y; and         X(28): I, V, T, L, R, F and M; and         X(30): T, P, Y, A, N, S, I, V, R, L, M, and H; and         X(31): T, S, P, N, M and I; and         X(32): A, N, T, S, D, R, FQ, P, I, E, V, M, L, K, H, C, and B;         and         X(33): V, A, L, M, G, R, and C; and         X(34): X; and         X(35): W, R, G, L, M, and P; and         X(36): N, S, D, B, K, E, R, Q, M, and G; and         X(37): S, T, A, N, D, V, I, E, Y, K, L, R, G, P, M, F, W, H, Q,         B, and C; and         X(38): S, T, N, I, G, R, L, C, A, W, M and E; and         X(39): W, G, A, R, E, C, Y, V, S, M, and H; and         X(40): X; and         X(41): N, G, K, S, D, E, T, R, H, P, A, B, V, Q, Y, M, and I;         and         X(42): K, R, N, D, S, T, G, E, I, V, Y, Q, P, H, A, W, M, and C,         and         X(43): S, T, N, K, I, R, D, E, P, L, A, W, G, M, H, Y, F, V, and         C,         X(44): L, Y, Q, F, E, H, S, V, K, M, T, I, W, N, D, R, P, A, and         G; and         X(45): D, E, N, S, T, K, G, L, A, Q, H, I, Y, B, R, V, P, M, F,         W, Z, and C; and         X(46): E, D, Q, Y, K, N, T, S, A, W, H, M, R, I, G, L, V, Z, F,         B, and P; and         X(47): I, D, E, M, G, T, Q, S, W, L, N, Y, K, V, R, F, A, P, and         H, and         X(48): W, I, T, N, D, E, L, G, S, Y, R, V, K, H, A, Q, M, and F;         and         X((49): D, N, E, G, W, Q, K, H, L, B, S, I, Y, T, A, R, M, Z,         and V; and         X(50): N, D, T, K, S, H, L, G, E, W, I, Q, M, R, B, Y, P, and A;

This consensus sequence contains 50 amino acids, in which the specific amino acids in position 10, 19, 24, 34 and 40 are defined as affecting the immunogenic properties of a HIV-1 envelope polypeptide, and the 45 remaining positions are randomly defined including the most common amino acids of wild-type HIV ENV proteins.

In fact, the teaching of WO 2010/022,740 is a transposition from endogenous retroviruses (ERV) or onco retroviruses to lentivirus on the basis of the teaching of WO 2005/095,442 (Inventors: Renard, Mangeney & Heidmann), but said transposition is inappropriate in the case of lentivirus, as shown by the Inventor of the present invention.

Briefly speaking, Dr Heidmann is an Inventor in WO 2005/095,442 and in the present invention. As a matter of fact, the effects of the mutations described in WO 2005/095,442 were also tested in lentivirus by the Inventor of the present invention, but no effect was observed when the mutations identified in endogenous retroviruses or onco retroviruses were transposed into ENV protein of lentivirus.

Moreover, since any amino acid can be assigned to the positions 10, 19, 24, 34 or 40 in the consensus sequence, WO 2010/022,740 teaches that such mutations can be effective using any amino acid residue. This teaching is in contradiction with the present invention, showing that the immunosuppressive properties of HIV-1 ENV protein are only affected by specific mutations that are defined not only by their position, but also by the nature of the substituted amino acid residues.

Furthermore, WO 2010/022,740 discloses experimental results for only one specific mutation within the immunosuppressive domain of HIV ENV protein, as defined by the 50 amino acids consensus sequence. The mutation, a substitution by R as the only one exemplified in the international application WO 2010/022,740, occurs at the amino acid in position 19, which again is equivalent to the position disclosed in the international application WO 2005/095,442 if one simply aligns ENV sequences (see FIG. 3 in WO 2010/022,740, which is a copy of FIG. 3 in Benit et al. 2001, Journal of Virology, Vol. 75, No. 23, p. 11709-11719) (Of note not only the position of the amino acid, but also the nature of the substitution (by arginine) is similar to the one described in WO 2005/095,442).

More specifically, when aligning the ENV sequences respectively of an endogenous retrovirus or onco retrovirus and a lentivirus, according to FIG. 3 of Benit et al.:

it appears that the position 19 in lentivirus corresponds to the position in WO 2005/095,442 where a specific substitution into arginine, E→R for endogenous or onco retrovirus, results in loss of immunosuppressive activity.

The international application WO 2010/022,740 discloses that said mutated HIV ENV protein inhibits proliferation of PBMC ex vivo, but such ex vivo result has no in vivo predictive value.

However, WO 2010/022,740 never discloses or suggests that mutants are efficient in vivo, i.e. that cell expressing mutants are detected by the immune system.

Moreover, as disclosed hereafter, such mutations are not efficient in vivo. Indeed, results disclosed hereafter in the example section relative to the present invention demonstrate that said substitution G19R does not inhibit in vivo the immunosuppressive properties of the ENV protein.

SUMMARY OF THE INVENTION

As a consequence, WO 2010/022,740 raises the same technical problem as the present invention, but does not offer an appropriate technical solution. This prior art reveals the difficulties to overcome the identification of the effective mutations affecting the immunosuppressive properties of the lentiviral ENV proteins.

Thus, the provision of in vivo effective non immunosuppressive lentiviral ENV proteins remains.

One aim of the invention is to provide new mutated ENV proteins devoid of immunosuppressive properties.

Another aim of the invention is to provide a new pharmaceutical composition efficient for treating lentiviral infection.

Another aim of the invention is to provide an efficient vaccine.

-   -   The invention relates to a pharmaceutical composition comprising         as active substance:         -   a) an isolated mutated human or simian lentiviral ENV             protein having substantially no immunosuppressive activity,         -   said mutated human or simian lentiviral ENV protein             resulting from mutation of the transmembrane (TM) subunit of             a wild type human or simian lentiviral ENV protein,         -   said mutated human or simian lentiviral ENV protein having             at least 70% identity, preferably at least 80% identity, to             one sequence chosen from the group consisting of SEQ ID NO:             216, SEQ ID NO: 420 and SEQ ID NO: 421,         -   said mutated human or simian lentiviral ENV protein             comprising a mutated immunosuppressive domain (ISU)             containing the following amino acid sequence:

(SEQ ID NO: 416) A-[IN]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

-   -   -   wherein,         -   X represents any amino acid,             -   and either         -   X_(a) is A, F, G, L, R or deleted, and X_(b) is C, D, E, H,             I, K, L, M, N, P, Q, S, T, V, W or Y, or         -   X_(a) is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and             X_(b) is A, F, G, R or deleted,         -   or         -   X_(a) is A, F, G, L, R or deleted, and X_(b) is A, F, G, R             or deleted,         -   or         -   b) at least one fragment of said isolated mutated human or             simian lentiviral ENV protein having substantially no             immunosuppressive activity,         -   said fragment comprising at least 40 amino acids,         -   said fragment comprising a mutated immunosuppressive domain             (ISU) containing the following amino acid sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

-   -   -   wherein,         -   X represents any amino acid,             -   and either         -   X_(a) is A, F, G, L, R or deleted, and X_(b) is C, D, E, H,             I, K, L, M, N, P, Q, S, T, V, W or Y, or         -   X_(a) is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and             X_(b) is A, F, G, R or deleted,         -   or         -   X_(a) is A, F, G, L, R or deleted, and X_(b) is A, F, G, R             or deleted,         -   in association with a pharmaceutically acceptable carrier,         -   said substantial absence of immunosuppressive activity of             the above mentioned mutated human or simian lentiviral ENV             protein or of the above defined fragment being liable to be             assessed by the fact that in an in vivo assay involving             engrafted tumor cells rejection,         -   said tumor cells being transduced either so as to express             said mutated ENV protein or said fragment (“mutated ENV             tumor cells”),         -   or said tumor cells being transduced so as to express said             wild type ENV protein or a fragment thereof (“wild type ENV             tumor cells”),         -   or said tumor cells being not transduced (“normal tumor             cells”),         -   the following ratio:         -   immunosuppression index of said mutated ENV protein or of             said fragment (i_(mutated env))/immunosuppression index of             wild type ENV protein (i_(wild type env)) is less than 0.5,         -   i_(mutated env) being defined by: (maximum area reached by             mutated ENV tumor cells−maximum area reached by normal tumor             cells)/(maximum area reached by normal tumor cells), and         -   i_(wild type env) being defined by: (maximum area reached by             wild type ENV tumor cells−maximum area reached by normal             tumor cells)/(maximum area reached by normal tumor cells).

The present application is based on the unexpected observation made by the Inventors that some specific amino acids of the immunosuppressive domain of a lentiviral ENV protein can be mutated conferring to said lentiviral ENV protein essentially no immunosuppressive properties, or no immunosuppressive properties, while retaining its antigenicity, the three-dimensional structure of the immunosuppressive domain, and its expression at the plasma membrane. Moreover, the mutated lentiviral ENV protein according to the invention does not alter the infectivity of a virus expressing it.

In the invention, the “mutated simian or human lentiviral ENV proteins” means that the ENV proteins derive from the expression of an env gene of a lentivirus of human or simian.

Lentiviruses according to the invention encompassed by the invention are HIV-1 and 2 and Simian immunodeficiency virus (SIV).

Because of the high mutation rate of HIV-1, HIV-2 and SIV viruses, the “mutated ENV protein”, as defined in the invention, encompasses two meanings.

According to the first meaning, the said “mutated ENV protein” is the unnatural result of the intervention of human beings.

According to the second meaning, the mutated ENV protein also encompasses naturally occurring variants for which up to now the non immunosuppressive properties remain unknown.

This second meaning takes into consideration the natural variability of HIV and SIV variants inside a same infected individual, wherein the said “mutated ENV protein” might be non immunosuppressive but its property is undetectable because an HIV infected patient always carries many HIV variants, the majority of which is immunosuppressive.

The three following proteins correspond to wild type sequences of the ENV protein of HIV-1, HIV-2 and SIV respectively. In the invention, they are considered as reference sequences of wild type ENV proteins.

SEQ ID NO: 417 wild type HIV-1 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL RAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCS GKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYTS LIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIK IFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPD RPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRLR DLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSL LNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 418 Wild type HIV-2 MCGRNQLFVASLLASACLIYCVQYVTVFYGVPVWRNASIPLFC ATKNRDTWGTIQCLPDNDDYQEIALNVTEAFDAWNNTVTEQA VEDVWSLFETSIKPCVKLTPLCVAMRCNSTTAKNTTSTPTTTTT ANTTIGENSSCIRTDNCTGLGEEEMVDCQFNMTGLERDKKKLY NETWYSKDVVCESNDTKKEKTCYMNHCNTSVITESCDKHYW DTMRFRYCAPPGFALLRCNDTNYSGFEPNCSKVVAATCTRMM ETQTSTWFGFNGTRAENRTYIYWHGRDNRTIISLNKFYNLTVH CKRPGNKTVVPITLMSGLVFHSQPINRRPRQAWCWFKGEWKE AMKEVKLTLAKHPRYKGTNDTEKIRFIAPGERSDPEVAYMWT NCRGEFLYCNMTWFLNWVENRTNQTQHNYVPCHIKQIINTWH KVGKNVYLPPREGQLTCNSTVTSIIANIDGGENQTNITFSAEVA ELYRLELGDYKLIEVTPIGFAPTPVKRYSSAPVRNKRGVFVLGF LGFLTTAGAAMGAASLTLSAQSRTLLAGIVQQQQQLLDVVKR QQEMLRLTVWGTKNLQARVTAIEKYLKDQAQLNSWGCAFRQ VCHTTVPWVNDTLTPDWNNMTWQEWEQRIRNLEANISESLEQ AQIQQEKNMYELQKLNSWDVFGNWFDLTSWIKYIQYGVYIVV GIIVLRIVIYVVQMLSRLRKGYRPVFSSPPAYFQQIHIHKDREQP AREETEEDVGNSVGDNWWPWPIRYIHFLIRQLIRLLNRLYNICR DLLSRSFQTLQLISQSLRRALTAVRDWLRFNTAYLQYGGEWIQ EAFRAFARATGETLTNAWRGFWGTLGQIGRGILAVPRRIRQGA EIALL SEQ ID NO: 419 wild type SIV mac239 MGCLGNQLLIAILLLSVYGIYCTLYVTVFYGVPAWRNATIPLFC ATKNRDTWGTTQCLPDNGDYSEVALNVTESFDAWNNTVTEQ AIEDVWQLFETSIKPCVKLSPLCITMRCNKSETDRWGLTKSITT TASTTSTTASAKVDMVNETSSCIAQDNCTGLEQEQMISCKFNM TGLKRDKKKEYNETWYSADLVCEQGNNTGNESRCYMNHCNT SVIQESCDKHYWDAIRFRYCAPPGYALLRCNDTNYSGFMPKCS KVVVSSCTRMMETQTSTWFGFNGTRAENRTYIYWHGRDNRTI ISLNKYYNLTMKCRRPGNKTVLPVTIMSGLVFHSQPINDRPKQ AWCWFGGKWKDAIKEVKQTIVKHPRYTGTNNTDKINLTAPGG GDPEVTFMWTNCRGEFLYCKMNWFLNWVEDRNTANQKPKE QHKRNYVPCHIRQIINTWHKVGKNVYLPPREGDLTCNSTVTSLI ANIDWIDGNQTNITMSAEVAELYRLELGDYKLVEITPIGLAPTD VKRYTTGGTSRNKRGVFVLGFLGFLATAGSAMGAASLTLTAQ SRTLLAGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTA IEKYLKDQAQLNAWGCAFRQVCHTTVPWPNASLTPKWNNET WQEWERKVDFLEENITALLEEAQIQQEKNMYELQKLNSWDVF GNWFDLASWIKYIQYGVYIVVGVILLRIVIYIVQMLAKLRQGY RPVFSSPPSYFQQTHIQQDPALPTREGKERDGGEGGGNSSWPW QIEYIHFLIRQLIRLLTWLFSNCRTLLSRVYQILQPILQRLSATLQ RIREVLRTELTYLQYGWSYFHEAVQAVWRSATETLAGAWGDL WETLRRGGRWILAIPRRIRQGLELTLL

Variant in the invention encompasses SIV, HIV-1 and HIV-2 ENV proteins.

Variants of the HIV-1 mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the wild type amino acid sequence of the HIV-1 ENV protein, and comprises the mutations as described above, and harbour no immunosuppressive activity.

Variants of the HIV-2 mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the wild type amino acid sequence of the HIV-2 ENV protein, and comprises the mutations as described above, and harbour no immunosuppressive activity.

Variants of the SIV mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the wild type amino acid sequence of the SIV ENV protein, and comprises the mutations as described above, and harbour no immunosuppressive activity.

The three following proteins (SEQ ID NO: 216, 420 and 421) correspond to three mutated sequences of the ENV protein of HIV-1, HIV-2 and SIV respectively. In the invention, they are considered as reference sequences of the mutated ENV proteins.

More specifically, SEQ ID NO: 216 corresponds to the SEQ ID NO: 417 in which the amino acid residue Y in position 5 (X_(a)) of the sequence A-[I/V]-E-[K/R]-X_(a)X_(b)-X-D-Q has been substituted by R.

SEQ ID NO: 420 corresponds to the SEQ ID NO: 418 in which the amino acid residue Y in position 5 (X_(a)) of the sequence A-[I/V]-E-[K/R]-X_(a)X_(b)-X-D-Q has been substituted by R.

SEQ ID NO: 421 corresponds to the SEQ ID NO: 419 in which the amino acid residue Y in position 5 (X_(a)) of the sequence A-[I/V]-E-[K/R]-X_(a)X_(b)-X-D-Q has been substituted by R.

SEQ ID NO: 216 mutated HIV-1 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL RAIEAQQHLLQLTVWGIKQLQARILAVERRLKDQQLLGIWGCS GKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYTS LIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIK IFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPD RPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRLR DLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSL LNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 420 mutated HIV-2 MCGRNQLFVASLLASACLIYCVQYVTVFYGVPVWRNASIPLFC ATKNRDTWGTIQCLPDNDDYQEIALNVTEAFDAWNNTVTEQA VEDVWSLFETSIKPCVKLTPLCVAMRCNSTTAKNTTSTPTTTTT ANTTIGENSSCIRTDNCTGLGEEEMVDCQFNMTGLERDKKKLY NETWYSKDVVCESNDTKKEKTCYMNHCNTSVITESCDKHYW DTMRFRYCAPPGFALLRCNDTNYSGFEPNCSKVVAATCTRMM ETQTSTWFGFNGTRAENRTYIYWHGRDNRTIISLNKFYNLTVH CKRPGNKTVVPITLMSGLVFHSQPINRRPRQAWCWFKGEWKE AMKEVKLTLAKHPRYKGTNDTEKIRFIAPGERSDPEVAYMWT NCRGEFLYCNMTWFLNWVENRTNQTQHNYVPCHIKQIINTWH KVGKNVYLPPREGQLTCNSTVTSIIANIDGGENQTNITFSAEVA ELYRLELGDYKLIEVTPIGFAPTPVKRYSSAPVRNKRGVFVLGF LGFLTTAGAAMGAASLTLSAQSRTLLAGIVQQQQQLLDVVKR QQEMLRLTVWGTKNLQARVTAIEKRLKDQAQLNSWGCAFRQ VCHTTVPWVNDTLTPDWNNMTWQEWEQRIRNLEANISESLEQ AQIQQEKNMYELQKLNSWDVFGNWFDLTSWIKYIQYGVYIVV GIIVLRIVIYVVQMLSRLRKGYRPVFSSPPAYFQQIHIHKDREQP AREETEEDVGNSVGDNWWPWPIRYIHFLIRQLIRLLNRLYNICR DLLSRSFQTLQLISQSLRRALTAVRDWLRFNTAYLQYGGEWIQ EAFRAFARATGETLTNAWRGFWGTLGQIGRGILAVPRRIRQGA EIALL SEQ ID NO: 421 mutated SIV mac239 MGCLGNQLLIAILLLSVYGIYCTLYVTVFYGVPAWRNATIPLFC ATKNRDTWGTTQCLPDNGDYSEVALNVTESFDAWNNTVTEQ AIEDVWQLFETSIKPCVKLSPLCITMRCNKSETDRWGLTKSITT TASTTSTTASAKVDMVNETSSCIAQDNCTGLEQEQMISCKFNM TGLKRDKKKEYNETWYSADLVCEQGNNTGNESRCYMNHCNT SVIQESCDKHYWDAIRFRYCAPPGYALLRCNDTNYSGFMPKCS KVVVSSCTRMMETQTSTWFGFNGTRAENRTYIYWHGRDNRTI ISLNKYYNLTMKCRRPGNKTVLPVTIMSGLVFHSQPINDRPKQ AWCWFGGKWKDAIKEVKQTIVKHPRYTGTNNTDKINLTAPGG GDPEVTFMWTNCRGEFLYCKMNWFLNWVEDRNTANQKPKE QHKRNYVPCHIRQIINTWHKVGKNVYLPPREGDLTCNSTVTSLI ANIDWIDGNQTNITMSAEVAELYRLELGDYKLVEITPIGLAPTD VKRYTTGGTSRNKRGVFVLGFLGFLATAGSAMGAASLTLTAQ SRTLLAGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTA IEKRLKDQAQLNAWGCAFRQVCHTTVPWPNASLTPKWNNET WQEWERKVDFLEENITALLEEAQIQQEKNMYELQKLNSWDVF GNWFDLASWIKYIQYGVYIVVGVILLRIVIYIVQMLAKLRQGY RPVFSSPPSYFQQTHIQQDPALPTREGKERDGGEGGGNSSWPW QIEYIHFLIRQLIRLLTWLFSNCRTLLSRVYQILQPILQRLSATLQ RIREVLRTELTYLQYGWSYFHEAVQAVWRSATETLAGAWGDL WETLRRGGRWILAIPRRIRQGLELTLL

The invention also encompasses the variants of the “mutated simian or human lentiviral ENV protein”, harbouring the above mentioned mutations, and conferring a lack of immunosuppressive properties to said variant.

Variants of the HIV-1 mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the reference mutated sequence of HIV-1 ENV protein (SEQ ID NO: 216), and comprises the mutations as described above, and harbour no immunosuppressive activity.

Variants of the HIV-2 mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the reference mutated sequence of the HIV-2 ENV protein (SEQ ID NO: 420), and comprises the mutations as described above, and harbour no immunosuppressive activity.

Variants of the SIV mutated ENV proteins according to the invention have at least 70%, preferably at least 80%, more preferably at least 90% of identity with the reference mutated sequence of the SIV ENV protein (SEQ ID NO: 421), and comprises the mutations as described above, and harbour no immunosuppressive activity.

The immunosuppressive domain (ISU) of the lentivirus according to the invention can be delimited by the sequence SEQ ID NO: 6,

(SEQ ID NO: 6) xGIVQQQxxLLxxxxxxQxxLxLxxWGxKxLQxRxxA[I/V]E[K/R] YLxDQxxLx in which x represents any amino acid.

SEQ ID NO: 6 corresponds to the non mutated ISU domain.

In this SEQ ID NO: 6, “x” (in small letters) is to be considered independently from “X” (in capital letters) used for the first time in SEQ ID NO: 416 of the present invention.

SEQ ID NO: 6 comprises SEQ ID NO: 1.

The most advantageous immunosuppressive domains of the wild type ENV proteins according to the invention comprise the following sequences:

HIV-1 ENV protein comprises the amino acid sequence AVERYLKDQ (SEQ ID NO: 7),

HIV-2 ENV protein comprises the amino acid sequence AIEKYLKDQ (SEQ ID NO: 8), and

SIV ENV protein comprises the amino acid sequence SEQ ID NO: 7 or 8.

When applying the mutations as defined above, the ISU domain looses its immunosuppressive properties.

In said SEQ ID NO: 6, the underlined amino acids are the essential amino acids.

In the invention, the ISU domain, as defined by SEQ ID NO: 6, is the minimal essential domain of the ENV protein according to the invention which harbours an immunosuppressive activity.

HIV-1 (SEQ ID NO: 366) SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLG (SEQ ID NO: 367) SGIVQQQNNLLRAIEAQQHLLKLTVWGIKQLQARILAVERYLKDQQLLG (SEQ ID NO: 368) SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLG (SEQ ID NO: 369) SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLG (SEQ ID NO: 370) SGIVQQQNNLLRAIEAQQQMLQLTVWGIKQLRARVLAVERYLRDQQLLG (SEQ ID NO: 371) SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERYLRDQQLLG SIV (SEQ ID NO: 372) SGIVQQQNNLLKAIEAQQHLLQLSIWGVKQLQARLLAVERYLQDQQILG (SEQ ID NO: 373) SGIVQQQNNLLRAIEAQQHLLQLSVWGIKQLQARVLAIERYLRDQQILG (SEQ ID NO: 374) AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVSAIEKYLKDQAQLN (SEQ ID NO: 375) AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKYLKDQAQLN HIV2 (SEQ ID NO: 376) AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKYLKDQAQLN (SEQ ID NO: 377) AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKYLKDQALLN

The localization of an ISU domain can be determined in all ENV proteins of viruses as described in Benit et al. 2001, Journal of Virology, Vol. 75, No. 23, p. 11709-11719. In a broad meaning, the ISU domain is defined by its structure and its localization, irrespective of the fact that it possesses or not an immunosuppressive activity.

In the invention, the ISU domain refers to a specific domain in which a mutation can affect the immunosuppressive property of the ENV protein.

The immunosuppressive property of the ENV protein is preferably measured using in vivo procedures, which are representative of the physiological environment.

The immunosuppressive properties of the mutated ENV proteins according to the invention are measured according to an in vivo procedure to assay the immunosuppressive activity of a ENV protein disclosed previously [Mangeney and Heidmann Proc Natl Acad Sci USA 1998; 95: 14920-14925; Mangeney et al. Proc Natl Acad Sci USA, 2007, 104(51):20534-9].

As a physiological test, this in vivo procedure is performed using ENV proteins, or fragment thereof, which are not associated to another component or carrier proteins, such as BSA.

Briefly, a wild-type (wild type lentiviral ENV protein) or modified nucleic acid expressing the protein to be tested (mutated lentiviral ENV protein) is transfected in tumour cell lines such as MCA 205 or C18.1 cell lines by known transfection methods. The tumour cells expressing the protein to be tested are then injected especially subcutaneous (s.c.) injection to a host, generally mice. Following said injection, the establishment of tumour or, to the contrary, its rejection, is determined and the tumour area is measured. Tumor establishment was determined by palpation and tumor area (mm²) was determined by measuring perpendicular tumor diameters. Immunosuppression index is defined as i=(S_(env)−S_(none))/S_(none), wherein S_(env) is the maximum area reached by a tumour expressing an envelope protein and S_(none) is the maximum area reached by a tumour not expressing ENV protein (negative control).

According to an embodiment of the invention, the above defined ratio relative to the immunosuppressive index can be less than 0.2, and can even have a negative value (see FIGS. 2 and 3).

In vitro assay could be carried out, using high doses of synthetic peptides but they are indirect and less convincing, since the expression “immunosuppressive” is relevant when applied to animals possessing a complete immune system and not to cell lines.

An additional difficulty for the functional characterization of an ISU domain relies on the fact that the ISU carried by the retroviral ENV proteins is a highly structured proteic domain, with trimer formation within the complete ENV proteins (Caffrey M., Biochimica et Biophysica Acta, 1536:116-122, 2001; Caffrey et al., The EMBO Journal, Vol. 17, No. 16, p. 4572-4584, 1998). Such structures are not naturally formed with ISU peptides of limited length, and this is most probably why most studies carried out with peptides provide irrelevant results and/or are dependent on specific coupling of the peptides to carrier proteins (such as BSA, e.g. Denner et al., Current Science, AIDS 1994, 8:1063-1072).

As mentioned above, the ENV proteins according to the invention are mutated. This mutation is made in vitro. Thus, the mutated ENV proteins according to the invention are isolated, and does not correspond to naturally occurring counterpart.

As mentioned above, the lentiviral mutated ENV proteins are substantially devoid of immunosuppressive properties. This means that the mutated ENV proteins according to the invention have no, or have reduced immunosuppressive properties with respect to the natural non mutated ENV protein from a virus of the same species. For instance, a mutated HIV ENV protein according to the invention has reduced immunosuppressive properties with respect to the wild type HIV ENV protein.

In the invention, the terms “substantially devoid of immunosuppressive properties” means that the mutated ENV proteins according to the invention have an immunosuppressive index less than about 0.2 [Mangeney and Heidmann Proc Natl Acad Sci USA 1998; 95: 14920-14925; Mangeney et al. Proc Natl Acad Sci USA, 2007, 104(51):20534-9].

In the invention, structures responsible for the antigenicity of the mutated lentiviral ENV protein are essentially preserved.

As intended herein, the expression “structures responsible for antigenicity” relates to structures of the protein which are liable to interact with components of the immune system such as antibodies or membrane receptors of immune cells, in particular T cells.

The mutation(s) within the immunosuppressive domain of the lentiviral ENV proteins is (are) sufficient to decrease the immunosuppressive activity of the mutated lentiviral ENV protein with respect to the corresponding wild type ENV. However, it might be advantageous that another amino acid be also mutated because it ensures that the structure of the mutated ENV protein is essentially conserved with respect to the corresponding wild type ENV protein.

The mutated lentiviral ENV protein has substantially retained the structure, especially the antigenic structure, e.g., immunogenic determinants, of the original determined lentiviral ENV protein, i.e. the wild type non mutated lentiviral ENV protein.

These properties can be evaluated by measuring the fusion and/or infectious properties of said mutated lentiviral ENV with respect to the same properties in the wild type non mutated lentiviral ENV protein (see example).

Generally speaking, the mutated ENV protein involved in the present invention has an average length of about 700 to about 950 amino acids.

The invention encompasses fragments of the mutated ENV protein as defined above, provided that said fragment:

-   -   comprises at least the sequence SEQ ID NO: 2, as defined above,     -   comprises at least 40 amino acids, preferably comprises at least         50 amino acids,     -   is substantially devoid of immunosuppressive properties, as         defined above,     -   preferably, comprises the extracellular parts of the ENV         protein,     -   retains the structure of the ENV protein from which it derives,     -   harbours the same epitopes as the corresponding fragment in the         wild type ENV protein.

According to a particular embodiment, the fragment of the mutated ENV protein of the invention can comprise about 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 700 amino acids. These values are given only in an illustrative way, as the man skilled in the art will understand that the fragment can comprise any number of amino acids comprised between 40 and 700.

Advantageously, the fragments according to the invention are such that, while retaining the antigenic structure of the full length mutated ENV protein, and thus of the wild type ENV protein, they have lost major antigenic regions that are responsible for antigenicity in another region than the region corresponding to the immunosuppressive domain.

The invention also relates to a pharmaceutical composition wherein the active substance is an isolated non naturally occurring mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, as above defined, or fragments thereof.

In a particular embodiment, the invention relates to a pharmaceutical composition as defined above comprising as active substance:

-   -   a) an isolated mutated human or simian lentiviral ENV protein         having substantially no immunosuppressive activity,     -   said mutated human or simian lentiviral ENV protein resulting         from mutation of the transmembrane (TM) subunit of a wild type         human or simian lentiviral ENV protein,     -   said mutated human or simian lentiviral ENV protein comprising a         mutated immunosuppressive domain (ISU) containing the following         amino acid sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

-   -   wherein,     -   X represents any amino acid,         -   and either     -   X_(a) is A, F, G, L or R, and X_(b) is C, D, E, H, I, K, L, M,         N, P, Q, S, T, V, W or Y, or     -   X_(a) is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and         X_(b) is A, F, G or R, or     -   X_(a) is A, F, G, L or R, and X_(b) is A, F, G or R,     -   or     -   b) at least one fragment of said isolated mutated human or         simian lentiviral ENV protein having substantially no         immunosuppressive activity,     -   said fragment comprising at least 40 amino acids,     -   said fragment comprising a mutated immunosuppressive domain         (ISU) containing the following amino acid sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

-   -   wherein,     -   X represents any amino acid,         -   and either     -   X_(a) is A, F, G, L or R, and X_(b) is C, D, E, H, I, K, L, M,         N, P, Q, S, T, V, W or Y, or     -   X_(a) is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and         X_(b) is A, F, G or R, or     -   X_(a) is A, F, G, L or R, and X_(b) is A, F, G or R,     -   in association with a pharmaceutically acceptable carrier.

The invention relates to a pharmaceutical composition as defined above comprising as active substance:

-   -   a) an isolated mutated human or simian lentiviral ENV protein         having substantially no immunosuppressive activity,     -   said mutated human or simian lentiviral ENV protein resulting         from mutation of the transmembrane (TM) subunit of a wild type         human or simian lentiviral ENV protein,     -   said mutated human or simian lentiviral ENV protein comprising a         mutated immunosuppressive domain (ISU) containing the following         amino acid sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

-   -   wherein     -   X represents any amino acid,         -   and either     -   X_(a) is A, F, G, L or R, and X_(b) is L, I, V, M or P, or     -   X_(a) is Y, I, H, C or T, and X_(b) is A, F, G or R, or     -   X_(a) is A, F, G, L or R, and X_(b) is A, F, G or R,     -   or     -   b) at least one fragment of said isolated mutated human or         simian lentiviral ENV protein having substantially no         immunosuppressive activity,     -   said fragment comprising at least 40 amino acids,     -   said fragment comprising a mutated immunosuppressive domain         (ISU) containing the following amino acid sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

-   -   wherein     -   X represents any amino acid,         -   and either     -   X_(a) is A, F, G, L or R, and X_(b) is L, I, V, M or P, or     -   X_(a) is Y, I, H, C or T, and X_(b) is A, F, G or R, or     -   X_(a) is A, F, G, L or R, and X_(b) is A, F, G or R,     -   in association with a pharmaceutically acceptable carrier.

According to a particular embodiment, the invention relates to a pharmaceutical composition as defined above comprising as active substance:

-   -   a) an isolated mutated human or simian lentiviral ENV protein         having substantially no immunosuppressive activity,     -   said mutated human or simian lentiviral ENV protein resulting         from mutation of the transmembrane (TM) subunit of a wild type         human or simian lentiviral ENV protein,     -   said mutated human or simian lentiviral ENV protein comprising a         mutated immunosuppressive domain (ISU) containing the following         amino acid sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

-   -   wherein     -   X represents any amino acid,         -   and either     -   X_(a) is A, G, or R, and X_(b) is C, D, E, H, I, K, L, M, N, P,         Q, S, T, V, W or Y, or     -   X_(a) is C, D, E, H, I, K, M, N, P, Q, 5, T, V, W or Y, and         X_(b) is F, G or R,     -   or     -   X_(a) is F or L, and X_(b) is F, G or R,     -   or     -   X_(a) is A, G or R, and X_(b) is A,     -   or     -   X_(a) is A, G or R, and X_(b) is F, G or R,     -   or     -   b) at least one fragment of said isolated mutated human or         simian lentiviral ENV protein having substantially no         immunosuppressive activity,     -   said fragment comprising at least 40 amino acids,     -   said fragment comprising a mutated immunosuppressive domain         (ISU) containing the following amino acid sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

-   -   wherein     -   X represents any amino acid,         -   and either     -   X_(a) is A, G, or R, and X_(b) is C, D, E, H, I, K, L, M, N, P,         Q, S, T, V, W or Y,     -   or     -   X_(a) is C, D, E, H, I, K, M, N, P, Q, 5, T, V, W or Y, and         X_(b) is F, G or R,     -   or     -   X_(a) is F or L, and X_(b) is F, G or R,     -   or     -   X_(a) is A, G or R, and X_(b) is A,     -   or     -   X_(a) is A, G or R, and X_(b) is F, G, R,     -   in association with a pharmaceutically acceptable carrier.

According to a particular embodiment, the invention relates to a pharmaceutical composition as defined above comprising as active substance:

-   -   a) an isolated mutated human or simian lentiviral ENV protein         having substantially no immunosuppressive activity,     -   said mutated human or simian lentiviral ENV protein resulting         from mutation of the transmembrane (TM) subunit of a wild type         human or simian lentiviral ENV protein,     -   said mutated human or simian lentiviral ENV protein comprising a         mutated immunosuppressive domain (ISU) containing the following         amino acid sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

-   -   wherein     -   X represents any amino acid,         -   and either     -   X_(a) is A, G, or R, and X_(b) is L, I, V, M or P,     -   or     -   X_(a) is Y, I, H, C or T, and X_(b) is F, G or R,     -   or     -   X_(a) is F or L, and X_(b) is F, G or R,     -   or     -   X_(a) is A, G or R, and X_(b) is A,     -   or     -   X_(a) is A, G or R, and X_(b) is F, G or R,     -   or     -   b) at least one fragment of said isolated mutated human or         simian lentiviral ENV protein having substantially no         immunosuppressive activity,     -   said fragment comprising at least 40 amino acids,     -   said fragment comprising a mutated immunosuppressive domain         (ISU) containing the following amino acid sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

-   -   wherein     -   X represents any amino acid,         -   and either     -   X_(a) is A, G, or R, and X_(b) is L, I, V, M or P,     -   or     -   X_(a) is Y, I, H, C or T, and X_(b) is F, G or R,     -   or     -   X_(a) is F or L, and X_(b) is F, G or R,     -   or     -   X_(a) is A, G or R, and X_(b) is A,     -   or     -   X_(a) is A, G or R, and X_(b) is F, G or R,     -   in association with a pharmaceutically acceptable carrier.

According to another particular embodiment, the invention also relates to a pharmaceutical composition as defined above comprising as active substance:

-   -   a) an isolated mutated human or simian lentiviral ENV protein         having substantially no immunosuppressive activity,     -   said mutated human or simian lentiviral ENV protein resulting         from mutation of the transmembrane (TM) subunit of a wild type         human or simian lentiviral ENV protein,     -   said mutated human or simian lentiviral ENV protein comprising a         mutated immunosuppressive domain (ISU) containing the following         amino acid sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

-   -   wherein,     -   X represents any amino acid,         -   and either     -   X_(a) is A, G, or R, and X_(b) is C, D, E, H, I, K, L, M, N, P,         Q, S, T, V, W or Y,     -   or     -   X_(a) is C, D, E, H, I, K, M, N, P, Q, 5, T, V, W or Y, and         X_(b) is F, G or R,     -   or     -   b) at least one fragment of said isolated mutated human or         simian lentiviral ENV protein having substantially no         immunosuppressive activity,     -   said fragment comprising at least 40 amino acids,     -   said fragment comprising a mutated immunosuppressive domain         (ISU) containing the following amino acid sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

-   -   wherein,     -   X represents any amino acid,         -   and either     -   X_(a) is A, G, or R, and X_(b) is C, D, E, H, I, K, L, M, N, P,         Q, S, T, V, W or Y,     -   or     -   X_(a) is C, D, E, H, I, K, M, N, P, Q, 5, T, V, W or Y, and         X_(b) is F, G or R,     -   in association with a pharmaceutically acceptable carrier.

According to another particular embodiment, the invention relates to a pharmaceutical composition as defined above, comprising as active substance:

-   -   a) an isolated mutated human or simian lentiviral ENV protein         having substantially no immunosuppressive activity,     -   said mutated human or simian lentiviral ENV protein resulting         from mutation of the transmembrane (TM) subunit of a wild type         human or simian lentiviral ENV protein,     -   said mutated human or simian lentiviral ENV protein comprising a         mutated immunosuppressive domain (ISU) containing the following         amino acid sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

-   -   wherein,     -   X represents any amino acid,         -   and either     -   X_(a) is A, G, or R, and X_(b) is L, I, V, M or P,     -   or     -   X_(a) is Y, I, H, C or T, and X_(b) is F, G or R,     -   or     -   b) at least one fragment of said isolated mutated human or         simian lentiviral ENV protein having substantially no         immunosuppressive activity,     -   said fragment comprising at least 40 amino acids,     -   said fragment comprising a mutated immunosuppressive domain         (ISU) containing the following amino acid sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

-   -   wherein,     -   X represents any amino acid,         -   and either     -   X_(a) is A, G, or R, and X_(b) is L, I, V, M or P,     -   or     -   X_(a) is Y, I, H, C or T, and X_(b) is F, G or R,     -   in association with a pharmaceutically acceptable carrier.     -   According to another particular embodiment, the invention         relates to a pharmaceutical composition as defined above,         -   wherein             -   X_(a) is R, and X_(b) is C, D, E, H, I, K, L, M, N, P,                 Q, S, T, V, W or Y, or             -   X_(a) is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y,                 and X_(b) is R, or             -   X_(a) is R, and X_(b) is R.

According to another particular embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the following amino acid sequences:

(SEQ ID NO: 422) A-I-E-K-X_(a)-X_(b)-X-DQ, (SEQ ID NO: 423) A-I-E-R-X_(a)-X_(b)-X-DQ, (SEQ ID NO: 424) A-V-E-K-X_(a)-X_(b)-X-DQ, (SEQ ID NO: 425) A-V-E-R-X_(a)-X_(b)-X-DQ.

In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein the resulting immunosuppressive domain, contained in the mutated lentiviral protein, comprises the amino acid sequence of the list consisting of: SEQ ID NO:9 to SEQ ID NO:37.

In the invention “SEQ ID NO: 9 to SEQ ID NO:37” encompass SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37.

The correspondence is the following one:

SEQ ID NO: 9 AVERALKD SEQ ID NO: 10 AVERFLKD SEQ ID NO: 11 AVERGLKD SEQ ID NO: 12 AVERLLKD SEQ ID NO: 13 AVERRLKD SEQ ID NO: 14 AVERYAKD SEQ ID NO: 15 AVERYFKD SEQ ID NO: 16 AVERYGKD SEQ ID NO: 17 AVERYRKD SEQ ID NO: 18 AVERAAKD SEQ ID NO: 19 AVERAFKD SEQ ID NO: 20 AVERAGKD SEQ ID NO: 21 AVERARKD SEQ ID NO: 22 AVERFAKD SEQ ID NO: 23 AVERFFKD SEQ ID NO: 24 AVERFGKD SEQ ID NO: 25 AVERFRKD SEQ ID NO: 26 AVERGAKD SEQ ID NO: 27 AVERGFKD SEQ ID NO: 28 AVERGGKD SEQ ID NO: 29 AVERGRKD SEQ ID NO: 30 AVERLAKD SEQ ID NO: 31 AVERLFKD SEQ ID NO: 32 AVERLGKD SEQ ID NO: 33 AVERLRKD SEQ ID NO: 34 AVERRAKD SEQ ID NO: 35 AVERRFKD SEQ ID NO: 36 AVERRGKD SEQ ID NO: 37 AVERRRKD

In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein the resulting immunosuppressive domain, contained in the mutated lentiviral protein, comprises the amino acid sequence of the list consisting of: SEQ ID NO:38 to SEQ ID NO:66.

In the invention “SEQ ID NO: 38 to SEQ ID NO:66” encompass SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66.

The correspondence is the following one:

SEQ ID NO: 38 AIEKALKD SEQ ID NO: 39 AIEKFLKD SEQ ID NO: 40 AIEKGLKD SEQ ID NO: 41 AIEKLLKD SEQ ID NO: 42 AIEKRLKD SEQ ID NO: 43 AIEKYAKD SEQ ID NO: 44 AIEKYFKD SEQ ID NO: 45 AIEKYGKD SEQ ID NO: 46 AIEKYRKD SEQ ID NO: 47 AIEKAAKD SEQ ID NO: 48 AIEKAFKD SEQ ID NO: 49 AIEKAGKD SEQ ID NO: 50 AIEKARKD SEQ ID NO: 51 AIEKFAKD SEQ ID NO: 52 AIEKFFKD SEQ ID NO: 53 AIEKFGKD SEQ ID NO: 54 AIEKFRKD SEQ ID NO: 55 AIEKGAKD SEQ ID NO: 56 AIEKGFKD SEQ ID NO: 57 AIEKGGKD SEQ ID NO: 58 AIEKGRKD SEQ ID NO: 59 AIEKLAKD SEQ ID NO: 60 AIEKLFKD SEQ ID NO: 61 AIEKLGKD SEQ ID NO: 62 AIEKLRKD SEQ ID NO: 63 AIEKRAKD SEQ ID NO: 64 AIEKRFKD SEQ ID NO: 65 AIEKRGKD SEQ ID NO: 66 AIEKRRKD

In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein the resulting immunosuppressive domain, contained in the mutated lentiviral protein, comprises the amino acid sequence of the list consisting of: SEQ ID NO: 67 to SEQ ID NO: 95.

In the invention “SEQ ID NO: 67 to SEQ ID NO:95” encompass SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94 and SEQ ID NO: 95.

The correspondence is the following one:

SEQ ID NO: 67 AIERALKD SEQ ID NO: 68 AIERFLKD SEQ ID NO: 69 AIERGLKD SEQ ID NO: 70 AIERLLKD SEQ ID NO: 71 AIERRLKD SEQ ID NO: 72 AIERYAKD SEQ ID NO: 73 AIERYFKD SEQ ID NO: 74 AIERYGKD SEQ ID NO: 75 AIERYRKD SEQ ID NO: 76 AIERAAKD SEQ ID NO: 77 AIERAFKD SEQ ID NO: 78 AIERAGKD SEQ ID NO: 79 AIERARKD SEQ ID NO: 80 AIERFAKD SEQ ID NO: 81 AIERFFKD SEQ ID NO: 82 AIERFGKD SEQ ID NO: 83 AIERFRKD SEQ ID NO: 84 AIERGAKD SEQ ID NO: 85 AIERGFKD SEQ ID NO: 86 AIERGGKD SEQ ID NO: 87 AIERGRKD SEQ ID NO: 88 AIERLAKD SEQ ID NO: 89 AIERLFKD SEQ ID NO: 90 AIERLGKD SEQ ID NO: 91 AIERLRKD SEQ ID NO: 92 AIERRAKD SEQ ID NO: 93 AIERRFKD SEQ ID NO: 94 AIERRGKD SEQ ID NO: 95 AIERRRKD

As mentioned above, the previous ENV proteins having their ISU comprising the above sequence are devoid of immunosuppressive properties.

Thus, in other words, any ENV protein of human or simian lentivirus having in their ISU an amino acid sequence comprising of the sequences SEQ ID NO: 9 to 95, is devoid of immunosuppressive properties.

In other words, a simian or human lentiviral ENV protein comprising, within its ISU domain, an amino acid sequence selected from SEQ ID NO: 9 to 95 is devoid of immunosuppressive properties.

In particular, the invention relates to a pharmaceutical composition as defined above, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the amino acid sequences:

-   -   SEQ ID NO: 13, SEQ ID NO: 42, SEQ ID NO: 71,     -   SEQ ID NO: 9 to 12,     -   SEQ ID NO: 14 to 41,     -   SEQ ID NO: 43 to 70, and     -   SEQ ID NO: 72 to 95.

In particular, the invention relates to a pharmaceutical composition as defined above, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the amino acid sequences:

-   -   SEQ ID NO: 13, SEQ ID NO: 42, SEQ ID NO: 71,     -   SEQ ID NO: 9, 11, 15 to 21, 23 to 29, 31 to 38, 40, 44 to 50, 52         to 58, 60 to 67, 69, 73 to 79, 81 to 87, 89 to 95.

In the invention, the fragments of the mutated ENV proteins according to the invention comprise or consist of the following sequences: SEQ ID NO: 67 to 211.

These fragments are also devoid of immunosuppressive properties.

However, these fragments retain the structure and the antigenicity of the corresponding immunosuppressive domain that is not mutated, i.e. the wild type immunosuppressive domain.

In the invention, the fragments of the mutated ENV proteins according to the invention comprise or consist of the following sequences: SEQ ID NO: 96 to 211.

The correspondences are as follows

SEQ ID NO: 96 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERAL KDQQLLG SEQ ID NO: 97 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERFL  KDQQLLG SEQ ID NO: 98 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERGL  KDQQLLG SEQ ID NO: 99 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERLL  KDQQLLG SEQ ID NO: 100 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERRL  KDQQLLG SEQ ID NO: 101 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERYA  KDQQLLG SEQ ID NO: 102 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERYF  KDQQLLG SEQ ID NO: 103 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERYG  KDQQLLG SEQ ID NO: 104 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERYR  KDQQLLG SEQ ID NO: 105 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERAA  KDQQLLG SEQ ID NO: 106 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERAF  KDQQLLG SEQ ID NO: 107 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERAG  KDQQLLG SEQ ID NO: 108 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERAR  KDQQLLG SEQ ID NO: 109 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERFA  KDQQLLG SEQ ID NO: 110 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERFF  KDQQLLG SEQ ID NO: 111 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERFG  KDQQLLG SEQ ID NO: 112 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERFR  KDQQLLG SEQ ID NO: 113 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERGA  KDQQLLG SEQ ID NO: 114 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERGF  KDQQLLG SEQ ID NO: 115 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERGG  KDQQLLG SEQ ID NO: 116 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERGR  KDQQLLG SEQ ID NO: 117 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERLA  KDQQLLG SEQ ID NO: 118 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERLF  KDQQLLG SEQ ID NO: 119 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERLG  KDQQLLG SEQ ID NO: 120 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERLR  KDQQLLG SEQ ID NO: 121 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERRA  KDQQLLG SEQ ID NO: 122 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERRF  KDQQLLG SEQ ID NO: 123 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERRG  KDQQLLG SEQ ID NO: 124 SGIVQQQSNLLRAIQARQHMLQLTVWGIKQLQARVLAVERRR  KDQQLLG SEQ ID NO: 125 AGIVQQQQQLLDVVKRQQLMLRLTVWGTKNLQARVTAIEKA  LKDQAQLNSWGCAFRQ SEQ ID NO: 126 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKF  LKDQAQLNSWGCAFRQ SEQ ID NO: 127 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKG  LKDQAQLNSWGCAFRQ SEQ ID NO: 128 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKL  LKDQAQLNSWGCAFRQ SEQ ID NO: 129 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKR  LKDQAQLNSWGCAFRQ SEQ ID NO: 130 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKY  AKDQAQLNSWGCAFRQ SEQ ID NO: 131 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKY  FKDQAQLNSWGCAFRQ SEQ ID NO: 132 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKY  GKDQAQLNSWGCAFRQ SEQ ID NO: 133 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKY  RKDQAQLNSWGCAFRQ SEQ ID NO: 134 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKA  AKDQAQLNSWGCAFRQ SEQ ID NO: 135 AGIVQQQQQLLDVVKRQQLMLRLTVWGTKNLQARVTAIEKA  FKDQAQLNSWGCAFRQ SEQ ID NO: 136 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKA  GKDQAQLNSWGCAFRQ SEQ ID NO: 137 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKA  RKDQAQLNSWGCAFRQ SEQ ID NO: 138 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKF  AKDQAQLNSWGCAFRQ SEQ ID NO: 139 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKF  FKDQAQLNSWGCAFRQ SEQ ID NO: 140 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKF  GKDQAQLNSWGCAFRQ SEQ ID NO: 141 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKF  RKDQAQLNSWGCAFRQ SEQ ID NO: 142 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKG AKDQAQLNSWGCAFRQ SEQ ID NO: 143 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKG  FKDQAQLNSWGCAFRQ SEQ ID NO: 144 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKG  GKDQAQLNSWGCAFRQ SEQ ID NO: 145 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKG  RKDQAQLNSWGCAFRQ SEQ ID NO: 146 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKL  AKDQAQLNSWGCAFRQ SEQ ID NO: 147 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKL  FKDQAQLNSWGCAFRQ SEQ ID NO: 148 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKL  GKDQAQLNSWGCAFRQ SEQ ID NO: 149 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKL  RKDQAQLNSWGCAFRQ SEQ ID NO: 150 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKR  AKDQAQLNSWGCAFRQ SEQ ID NO: 151 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKR  FKDQAQLNSWGCAFRQ SEQ ID NO: 152 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKR  GKDQAQLNSWGCAFRQ SEQ ID NO: 153 AGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKR  RKDQAQLNSWGCAFRQ SEQ ID NO: 154 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKA  LQDQARLNSWGCAFRQ SEQ ID NO: 155 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKF  LQDQARLNSWGCAFRQ SEQ ID NO: 156 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKG  LQDQARLNSWGCAFRQ SEQ ID NO: 157 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKL  LQDQARLNSWGCAFRQ SEQ ID NO: 158 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKR  LQDQARLNSWGCAFRQ SEQ ID NO: 159 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKY  AQDQARLNSWGCAFRQ SEQ ID NO: 160 AGIVQQQQQLLDVVKRQ0ELLRLTVWGTKNL0ARVTAIEKY  FQDQARLNSWGCAFRQ SEQ ID NO: 161 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKY  GQDQARLNSWGCAFRQ SEQ ID NO: 162 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKY  RQDQARLNSWGCAFRQ SEQ ID NO: 163 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKA  AQDQARLNSWGCAFRQ SEQ ID NO: 164 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKA  FQDQARLNSWGCAFRQ SEQ ID NO: 165 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKA  GQDQARLNSWGCAFRQ SEQ ID NO: 166 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKA  RQDQARLNSWGCAFRQ SEQ ID NO: 167 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKF  AQDQARLNSWGCAFRQ SEQ ID NO: 168 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKF  FQDQARLNSWGCAFRQ SEQ ID NO: 169 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKF  GQDQARLNSWGCAFRQ SEQ ID NO: 170 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKF  RQDQARLNSWGCAFRQ SEQ ID NO: 171 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKG  AQDQARLNSWGCAFRQ SEQ ID NO: 172 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKG  FQDQARLNSWGCAFRQ SEQ ID NO: 173 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKG  GQDQARLNSWGCAFRQ SEQ ID NO: 174 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKG  RQDQARLNSWGCAFRQ SEQ ID NO: 175 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKL  AQDQARLNSWGCAFRQ SEQ ID NO: 176 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKL  FQDQARLNSWGCAFRQ SEQ ID NO: 177 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKL  GQDQARLNSWGCAFRQ SEQ ID NO: 178 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKL  RQDQARLNSWGCAFRQ SEQ ID NO: 179 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKR  AQDQARLNSWGCAFRQ SEQ ID NO: 180 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKR  FQDQARLNSWGCAFRQ SEQ ID NO: 181 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKR  GQDQARLNSWGCAFRQ SEQ ID NO: 182 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKR  RQDQARLNSWGCAFRQ SEQ ID NO: 183 AGIVQOQQQLLDVVKROQELLRLTVWGTKNLQTRVTAIEKA  LKDQAQLNAWGCAFRQ SEQ ID NO: 184 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKF  LKDQAQLNAWGCAFRQ SEQ ID NO: 185 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKG  LKDQAQLNAWGCAFRQ SEQ ID NO: 186 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKL  LKDQAQLNAWGCAFRQ SEQ ID NO: 187 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKR  LKDQAQLNAWGCAFRQ SEQ ID NO: 188 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKY  AKDQAQLNAWGCAFRQ SEQ ID NO: 189 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKY  FKDQAQLNAWGCAFRQ SEQ ID NO: 190 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKY  GKDQAQLNAWGCAFRQ SEQ ID NO: 191 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKY  RKDQAQLNAWGCAFRQ SEQ ID NO: 192 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKA  AKDQAQLNAWGCAFRQ SEQ ID NO: 193 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKA  FKDQAQLNAWGCAFRQ SEQ ID NO: 194 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKA  GKDQAQLNAWGCAFRQ SEQ ID NO: 195 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKA  RKDQAQLNAWGCAFRQ SEQ ID NO: 196 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKF  AKDQAQLNAWGCAFRQ SEQ ID NO: 197 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKF  FKDQAQLNAWGCAFRQ SEQ ID NO: 198 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKF  GKDQAQLNAWGCAFRQ SEQ ID NO: 199 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAOEKF  RKDQAQLNAWGCAFRQ SEQ ID NO: 200 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKG  AKDQAQLNAWGCAFRQ SEQ ID NO: 201 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKG  FKDQAQLNAWGCAFRQ SEQ ID NO: 202 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKG  GKDQAQLNAWGCAFRQ SEQ ID NO: 203 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKG  RKDQAQLNAWGCAFRQ SEQ ID NO: 204 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKL  AKDQAQLNAWGCAFRQ SEQ ID NO: 205 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKL  FKDQAQLNAWGCAFRQ SEQ ID NO: 206 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKL GKDQAQLNAWGCAFRQ SEQ ID NO: 207 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKL  RKDQAQLNAWGCAFRQ SEQ ID NO: 208 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKR  AKDQAQLNAWGCAFRQ SEQ ID NO: 209 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKR  FKDQAQLNAWGCAFRQ SEQ ID NO: 210 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKR  GKDQAQLNAWGCAFRQ SEQ ID NO: 211 AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIEKR  RKDQAQLNAWGCAFRQ

In particular, the invention relates to a pharmaceutical composition as defined above, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the amino acid sequences: SEQ ID NO: 96, 98, 100, 102 to 108, 110 to 116, 118 to 125, 127, 129, 131 to 137, 139 to 145, 147 to 154, 156, 158, 160 to 166, 168 to 174, 176 to 183, 185, 187, 189 to 195, 197 to 203, 205 to 211.

Advantageously, the invention relates to the pharmaceutical composition as defined above, wherein said mutated protein consists of one of the following sequences SEQ ID NO: 212 to 269

HIV-1 LAI SEQ ID NO: 212 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNKKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERALKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFMTNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 213 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERALKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 214 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERGLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 215 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERLLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 216 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQFILLQLTVWGIKQLQARIL AVERRLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 217 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERYAKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSILIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE WQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 218 MRVKKKYQHLWRWGWKWGTMLLGIMLMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERYFKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 219 MRVKRKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISIAVDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERYGKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 220 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERYRKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 221 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERAAKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 222 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERAFKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 223 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERAGKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 224 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERARKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 225 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERFAKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 226 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERFFKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 227 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERFGKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 228 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERFRKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSKIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 229 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERGAKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 230 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERGFKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 231 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERGGKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 232 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERGRKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 233 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERLAKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 234 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERLFKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 235 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERLGKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 236 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERLRKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 237 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERRAKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 238 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERRFKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 239 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERRGKDQQLLGIWGCSGKLICTFAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 240 MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVY YGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDP NPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPC VKLTPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIK NCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTS VITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIV QLNQSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQA HCNISRAKWNATLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTIT LPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDG GNNNNGSEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT KAKRRVVQREKRAVGIGALFLGFLGAAGSTMGARSMTLTV QARQLLSGIVOQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL AVERRRKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQI WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIV NRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRL VNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGW EALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE VVQGACRAIRHIPRRIRQGLERILL HIV-BH10 SEQ ID NO: 241 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERA LKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 242 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERF LKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 243 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERG LKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 244 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERL LKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 245 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERR LKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 246 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERY AKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 247 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERY FKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMT WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASL WNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVRQ GYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSLA LIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYW WNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGAY RAIRHIPRRIRQGLERILL SEQ ID NO: 248 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERY GKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 249 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERY RKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 250 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV  YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERA AKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 251 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERA FKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMT WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASL WNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVRQ GYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSLA LIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYW WNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGAY RAIRHIPRRIRQGLERILL SEQ ID NO: 252 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERA GKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 253 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV  YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERA RKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 254 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERF AKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 255 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERF FKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMT WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASL WNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVRQ GYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSLA LIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYW WNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGAY RAIRHIPRRIRQGLERILL SEQ ID NO: 256 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSEEPIPIHYCAPAGFAILKCNNKTENGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQEGNNKTIIEKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERF GKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 257 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSEEPIPIHYCAPAGFAILKCNNKTENGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQEGNNKTIIEKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERF RKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 258 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSEEPIPIHYCAPAGFAILKCNNKTENGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQEGNNKTIIEKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERG AKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 259 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERG FKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMT WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASL WNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVRQ GYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSLA LIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYW WNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGAY RAIRHIPRRIRQGLERILL SEQ ID NO: 260 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERG GKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 261 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERG RKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 262 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERL AKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 263 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERL FKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMT WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASL WNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVRQ GYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSLA LIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYW WNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGAY RAIRHIPRRIRQGLERILL SEQ ID NO: 264 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERL GKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 265 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQEGNNKTIIEKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERL RKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 266 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSEEPIPIHYCAPAGFAILKCNNKTENGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQEGNNKTIIEKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERR AKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 267 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSEEPIPIHYCAPAGFAILKCNNKTENGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQEGNNKTIIEKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERR FKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMT WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASL WNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVRQ GYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSLA LIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYW WNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGAY RAIRHIPRRIRQGLERILL SEQ ID NO: 268 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERR GKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL SEQ ID NO: 269 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTV YYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTD PNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKP CVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQAC PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQS VEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS RAKWNNTLKQIDSKLREQFGNNKTIIFKQSSGGDPEIVTHSFN CGGEFFYCNSTQLFNSTWFNSTWSTKGSNNTEGSDTITLPCRI KQIINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSN NESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKR RVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQL LSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERR RKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNM TWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS LWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSVVNRVR QGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSL ALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKY WWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQG AYRAIRHIPRRIRQGLERILL

The invention also encompasses the variants of the above sequences, harbouring the above mentioned mutations, and conferring to said variant a lack of immunosuppressive properties.

In particular, the invention relates to a pharmaceutical composition as defined above, wherein said isolated mutated human or simian lentiviral ENV protein consists of one of the amino acid sequences: SEQ ID NO: 212, 214, 216, 218 to 224, 226 to 232, 234 to 241, 243, 245, 247 to 253, 255 to 261, 263 to 269.

The invention also relates to a pharmaceutical composition as defined above, wherein said mutated protein comprises additional mutations either downstream the C-terminal end of the sequence SEQ ID NO: 416 or upstream the N-terminal end of SEQ ID NO: 416.

These additional mutations can be advantageous to maintain the three-dimensional structure of the immunosuppressive domain and of the ENV protein (see details concerning the membrane expression of the ENV protein [see FIG. 4 and example] and concerning infectivity [see FIG. 5 and example]).

In a particular embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated protein comprises an additional mutation in one at least of the amino acids at positions 29, 36 and 37 of SEQ ID NO: 426:

(SEQ ID NO: 426) A[I/V]E[K/R]X_(a)X_(b)X₁DQX₂X₃LX₄X₅WGC[A/S][F/G]X₆X₇CVX₈ TX₉VPX_(c)X₁₀Z₁Z₂Z₃Z₄Z₅X_(d)X_(e)[S/T] wherein

-   -   X_(a) and X_(b) are as defined above,     -   X₁ to X₁₀ represent any amino acid,     -   Z₁ to Z₅ represent no amino acid or any amino acid,         independently from each other such that         -   X_(c) is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,             V, Y, or deleted, preferably A, D, or N,         -   X_(d) is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V,             Y, W, or deleted, preferably A, G, S or Y,         -   X_(e) is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V,             Y, W, or deleted, preferably A, D or N.

This sequence contains the following amino acid sequence A[I/V]E[K/R]X_(a)X_(b)X₁DQ (SEQ ID NO: 416) elongated on its C-terminal end in which additional mutations are present. It is to be noted that “X₁” in the above mentioned sequence SEQ ID NO: 426 has the same meaning as “X” in SEQ ID NO: 416.

In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, said mutated protein comprising the amino acid sequences of the group consisting of SEQ ID NO: 271 to SEQ ID NO: 283.

SEQ ID NO: 271, contains the mutations Y41R, K72A: SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVER R LKDQQLLGI WGCSGKLICTTAVPWNASWSN A SLEQIWNNMTWMEWDREINNYTSLIHSL IEESQNQQEKNEQEL. SEQ ID NO: 272, contains the mutations Y41R, K72G: SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVER R LKDQQLLGI WGCSGKLICTTAVPWNASWSN G SLEQIWNNMTWMEWDREINNYTSLIHSL IEESQNQQEKNEQEL. SEQ ID NO: 273, contains the mutations Y41R, K72S: SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVER R LKDQQLLGI WGCSGKLICTTAVPWNASWSN S SLEQIWNNMTWMEWDREINNYTSLIHSL IEESQNQQEKNEQEL. SEQ ID NO: 274, contains the mutations Y41R, W65D: SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVER R LKDQQLLGI WGCSGKLICTTAVP D NASWSNKSLEQIWNNMTWMEWDREINNYTSLIHSL IEESQNQQEKNEQEL. SEQ ID NO: 275, contains the mutations Y41R, W65A: SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVER R LKDQQLLGI WGCSGKLIVTTAVP A NASWSNKSLEQIWNNMTWMEWDREINNYTSLIHSL IEESQNQQEKNEQEL. SEQ ID NO: 276, contains the mutations Y41R, W65N: SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVER R LKDQQLLGI WGCSGKLICTTAVP N NASWSNKSLEQIWNNMTWMEWDREINNYTSLIHSL IEESQNQQEKNEQEL. SEQ ID NO: 277, contains the mutations Y41R, S73D: SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVER R LKDQQLLGI WGCSGKLICTTAVPWNASWSNK D LEQIWNNMTWMEWDREINNYTSLIHSL IEESQNQQEKNEQEL. SEQ ID NO: 278, contains the mutations Y41R, S73A: SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVER R LKDQQLLGI WGCSGKLICTTAVPWNASWSNK A LEQIWNNMTWMEWDREINNYTSLIHSL IEESQNQQEKNEQEL. SEQ ID NO: 279, contains the mutations Y41R, S73N: SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVER R LKDQQLLGI WGCSGKLICTTAVPWNASWSNK N LEQIWNNMTWMEWDREINNYTSLIHSL IEESQNQQEKNEQEL. SEQ ID NO: 280, contains the mutations Y41R, K72Y: SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVER R LKDQQLLGI WGCSGKLICTTAVPWNASWSN Y SLEQIWNNMTWMEWDREINNYTSLIHSL IEESQNQQEKNEQEL. SEQ ID NO: 281, contains the mutations R40A, Y41R, K72A: SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVE AR LKDQQLLGI WGCSGKLICTTAVPWNASWSN A SLEQIWNNMTWMEWDREINNYTSLIHSL IEESQNQQEKNEQEL. SEQ ID NO: 282, contains the mutations Y41R, K72A, S73A: SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVER R LKDQQLLGI WGCSGKLICTTAVPWNASWSN AA LEQIWNNMTWMEWDREINNYTSLIHSL IEESQNQQEKNEQEL. SEQ ID NO: 283, contains the mutations Y41R, W65A, K72A, S73A: SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVER R LKDQQLLGI WGCSGKLICTTAVP A NASWSN AA LEQIWNNMTWMEWDREINNYTSLIHSL IEESQNQQEKNEQEL.

The mutations in the above mutated peptides are indicated by the amino acid residues which are bolded and underlined.

In another aspect, the invention relates to a pharmaceutical composition as defined above, comprising an additional mutation of one at least of the amino acids X₁₁, X₁₂, X₁₃ and X₁₄ in the following sequence (containing the above defined SEQ ID NO: 416 sequence):

(SEQ ID NO: 428) X₁₁X₁₂ X₁₃X₁₄I LA[I/V]E[K/R]X_(a)X_(b)X₁DQ, wherein X₁ represents any amino acid, Xa and Xb are as defined above, X₁₁ is:

-   -   either deleted,     -   or A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, Y or W, in         particular A, G or R, and/or         X₁₂ is:     -   either deleted,     -   or A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, Y or W, in         particular A, G or R, and/or         X₁₃ is:     -   either deleted,     -   or C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y or W, in         particular R or G, and/or         X₁₄ is:     -   either deleted,     -   or A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, Y or W, in         particular A or G.

This sequence contains the following amino acid sequence A[I/V]E[K/R]X_(a)X_(b)X₁DQ (SEQ ID NO: 416) elongated on its N-terminal end in which additional mutations are present. It is to be noted that “X₁” in SEQ ID NO: 428 has exactly the same meaning as “X” in SEQ ID NO: 416.

In another advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated lentiviral protein, said variant or said fragment harbour a three-dimensional structure similar to the structure of the natural non mutated lentiviral ENV protein, non mutated lentiviral ENV variant or non mutated lentiviral ENV fragment thereof.

The skilled person knows how to measure the antigenicity, by using standard proceedings.

In another advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated lentiviral protein, said variant or said fragment are expressed at the plasma membrane at a level substantially identical to the expression at the plasma membrane of the natural non mutated lentiviral ENV protein, non mutated lentiviral ENV variant or non mutated lentiviral ENV fragment thereof.

The membrane expression of the lentiviral ENV protein according to the invention can be measured by any techniques allowing determination of a plasma membrane protein. For instance, cells can be transfected with an expression vector allowing the expression of the mutated lentiviral ENV protein according to the invention. Cells are then incubated with an antibody recognizing specifically the extracellular part of said lentiviral mutated ENV protein. The complex (antibody/ENV protein) is detected by another antibody, and the complex can be quantified by flow cytometry (see for instance FIG. 4, and example).

If no complex is detected, the mutated ENV protein is not expressed at the plasma membrane. On the contrary, if the protein is expressed, this means that the mutated ENV protein is expressed at the plasma membrane.

In another advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated lentiviral ENV protein is such that a lentivirus, or a pseudotype, expressing said mutated lentiviral ENV protein, instead of the non mutated ENV protein, has a viral titer similar to the viral titer of said lentivirus, or pseudotype, expressing the non mutated lentiviral protein.

Viral titer is measured according to a commonly used protocol, and as described in the example (see FIG. 5).

In another advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated lentiviral ENV protein is a HIV-1 lentiviral protein consisting of the amino acid sequences SEQ ID NO: 284 to SEQ ID NO: 292.

SEQ ID NO: 284 Y41R MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL RAIEAQQHLLQLTVWGIKQLQARILAVERRLKDQQLLGIWGC SGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYT SLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI KIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP DRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRL RDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVS LLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 285 Y41R, K72A MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL RAIEAQQHLLQLTVWGIKQLQARILAVERRLKDQQLLGIWGC SGKLICTTAVPWNASWSNASLEQIWNNMTWMEWDREINNYT SLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI KIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP DRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRL RDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVS LLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 286  Y41R, K72G MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL RAIEAQQHLLQLTVWGIKQLQARILAVERRLKDQQLLGIWGC SGKLICTTAVPWNASWSNGSLEQIWNNMTWMEWDREINNYT SLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI KIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP DRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRL RDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVS LLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 287 Y41R, K72S MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL RAIEAQQHLLQLTVWGIKQLQARILAVERRLKDQQLLGIWGC SGKLICTTAVPWNASWSNSSLEQIWNNMTWMEWDREINNYTS LIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIK IFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPD RPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRLR DLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSL LNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 288 Y41G MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL RAIEAQQHLLQLTVWGIKQLQARILAVERGLKDQQLLGIWGC SGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYT SLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI KIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP DRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRL RDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVS LLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 289 Y41L MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL RAIEAQQHLLQLTVWGIKQLQARILAVERLLKDQQLLGIWGC SGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYT SLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI KIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP DRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRL RDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVS LLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 290 Y41A MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL RAIEAQQHLLQLTVWGIKQLQARILAVERALKDQQLLGIWGC SGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYT SLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI KIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP DRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRL RDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVS LLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 291 Y41F MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL RAIEAQQHLLQLTVWGIKQLQARILAVERFLKDQQLLGIWGC SGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYT SLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI KIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP DRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRL RDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVS LLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL SEQ ID NO: 292 L42R MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYY GVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNP QEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKL TPLCVSLKCTDLGNATNTNSSNTNSSSGEMMMEKGEIKNCSFN ISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYTLTSCNTSVITQACP KVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI RPVVSTQLLLNGSLAEEEVVIRSANFTDNAKTIIVQLNQSVEINC TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWN ATLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFY CNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQFINMWQ EVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGG GDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAV GIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLL RAIEAQQHLLQLTVWGIKQLQARILAVERYRKDQQLLGIWGC SGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYT SLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI KIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP DRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRL RDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVS LLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL

Also, the following proteins are advantageous in the invention:

SEQ ID NO: 293 SIV MM251 L42R MGCLGNQLLIAILLLSVYGIYCTQYVTVFYGVPAWRNATIPLFC ATKNRDTWGTTQCLPDNGDYSELALNVTESFDAWENTVTEQA IEDVWQLFETSIKPCVKLSPLCITMRCNKSETDRWGLTKSSTTIT TAAPTSAPVSEKIDMVNETSSCIAQNNCTGLEQEQMISCKFTMT GLKRDKTKEYNETWYSTDLVCEQGNSTDNESRCYMNHCNTS VIQESCDKHYWDTIRFRYCAPPGYALLRCNDTNYSGFMPKCSK VVVSSCTRMMETQTSTWFGFNGTRAENRTYIYWHGRDNRTIIS LNKYYNLTMKCRRPGNKTVLPVTIMSGLVFHSQPINDRPKQA WCWFGGKWKDAIKEVKQTIVKHPRYTGTNNTDKINLTAPGGG DPEVTFMWTNCRGEFLYCKMNWFLNWVEDRDVTTQRPKERH RRNYVPCHIRQIINTWHKVGKNVYLPPREGDLTCNSTVTSLIAN IDWTDGNQTSITMSAEVAELYRLELGDYKLVEITPIGLAPTDVK RYTTGGTSRNKRGVFVLGFLGFLATAGSAMGAASLTLTAQSR TLLAGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVTAIE KYRKDQAQLNAWGCAFRQVCHTTVPWPNASLTPDWNNDTW QEWERKVDFLEENITALLEEAQIQQEKNMYELQKLNSWDVFG NWFDLASWIKYIQYGIYVVVGVILLRIVIYIVQMLAKLRQGYRP VFSSPPSYFQXTHTQQDPALPTREGKEGDGGEGGGNSSWPWQI EYIHFLIRQLIRLLTWLFSNCRTLLSRAYQILQPILQRLSATLRRV REVLRTELTYLQYGWSYFHEAVQAGWRSATETLAGAWRDLW ETLRRGGRWILAIPRRIRQGLELTLL SEQ ID NO: 294 HIV2 L42R MEPGRNQLFVVILLTSACLVYCSQYVTVFYGIPAWKNASIPLFC ATKNRDTWGTIQCLPDNDDYQEIILNVTEAFDAWNNTVTEQA VEDVWHLFETSIKPCVKLTPLCVAMNCSRVQGNTTTPNPRTSS STTSRPPTSAASIINETSNCIENNTCAGLGYEEMMQCEFNMKGL EQDKKRRYKDTWYLEDVVCDNTTAGTCYMRHCNTSIIKESCD KHYWDAMRFRYCAPPGFALLRCNDTNYSGFEPKCTKVVAASC TRMMETQTSTWFGFNGTRAENRTYIYWHGRDNRTIISLNKYY NLTMRCKRPGNKTVLPITLMSGLVFHSQPINTRPRQAWCRFGG RWREAMQEVKQTLVQHPRYKGINDTGKINFTKPGAGSDPEVA FMWTNCRGEFLYCNMTWFLNWVEDKNQTRRNYCHIKQIINT WHKVGKNVYLPPREGELACESTVTSIIANIDIDKNRTHTNITFS AEVAELYRLELGDYKLIEITPIGFAPTDQRRYSSTPVRNKRGVF VLGFLGFLATAGSAMGARSLTLSAQSRTLLAGIVQQQQQLLDV VKRQQEMLRLTVWGTKNLQARVTAIEKYRKHQAQLNSWGC AFRQVCHTTVPWVNDSLSPDWKNMTWQEWEKQVRYLEANIS QSLEEAQIQQEKNMYELQKLNSWDILGNWFDLTSWVKYIQYG VHIVVGIIALRIAIYVVQLLSRFRKGYRPVFSSPPGYLQQIHIHK DRGQPANEGTEEDVGGDSGYDLWPWPINYVQFLIHLLTRLLIG LYNICRDLLSKNSPTRRLISQSLTAIRDWLRLKAAQLQYGCEWI QEAFQAFARTTRETLAGAWGWLWEAARRIGRGILAVPRRIRQ GAELALL

Advantageously: the mutated lentiviral proteins are

1—SEQ ID NO: 284 to 294; these mutated ENV proteins having substantially no immunosuppressive properties advantageously,

2—SEQ ID NO: 284 to SEQ ID NO: 292; these mutated ENV proteins having substantially no immunosuppressive properties and being highly expressed at the plasma membrane, more advantageously,

3—SEQ ID NO: 284 to SEQ ID NO: 291; these mutated ENV proteins having substantially no immunosuppressive properties, being highly expressed at the plasma membrane and conferring to a virus expressing them a medium or high viral titer, in particular

4—Y41F (SEQ ID NO: 291), Y41L (SEQ ID NO: 289), Y41A (SEQ ID NO: 290); these mutated ENV proteins having substantially no immunosuppressive properties, being highly expressed at the plasma membrane and confers to a virus expressing them a high viral titer. As mentioned above “conferring to a virus expressing them a medium or high viral titer” means that, when the sequence of the wild type ENV protein is substituted by the sequence of the mutated ENV in the lentiviral or pseudotype retrovirus, the virus thus expresses a mutated lentiviral ENV protein, along with other wild type viral proteins (GAG, PRO, POL), said mutated ENV protein conferring to the virus the ability to enter in its target cell:

-   -   either at a level comparable or higher to the ability of the         wild type virus, i.e. high ability     -   or at a lower level compared to the ability of the wild type         virus, i.e. medium or low ability.

The ability to enter in the target cell can be measured by the viral load. Viral load is a measure of the amount of target cells that can be infected by a given amount (1 mL) of virus (see FIG. 5 and example).

The invention also relates to a pharmaceutical composition comprising a nucleic acid molecule coding for a mutated lentiviral ENV protein, or variant of said protein, or fragments thereof, as defined above, in association with a pharmaceutically acceptable carrier.

In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said nucleic acid is comprised in a vector, said vector comprising means allowing the expression of said mutated lentiviral ENV protein, or variant of said protein, or fragments thereof.

In another advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said nucleic acid is comprised in a vector, said nucleic acid being placed under the control of sequences that allow the expression of said mutated lentiviral ENV protein, or variant of said protein, or fragments thereof.

In another embodiment, the invention relates to a pharmaceutical composition, as defined above, in particular as a vaccine, comprising a DNA molecule coding for said mutated lentiviral ENV protein, or variant of said protein, or fragments thereof.

DNA vaccines expressing ENV proteins can be produced as described in Bellier et al., (DNA vaccines expressing retrovirus-like particles are efficient immunogens to induce neutralizing antibodies, Vaccine, 27(42):5772-80, 2009).

In another advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, said vector being chosen among an eukaryotic or prokaryotic expression vector, in particular an eukaryotic vector which is a viral vector, in particular a pox vector, such as a fowlpox, a canarypox, or a MVA (modified vaccinia virus Ankara) vector, an adenoviral vector, a lentiviral vector, a measles vector, a Sendai virus or a CMV (cytomegalovirus) vector.

In another advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, further comprising at least one nucleotide molecule coding for a GAG and/or a PRO and/or a POL protein and/or a mutated NEF protein substantially devoid of immunosuppressive properties, of a lentivirus, preferably a lentivirus of the same origin as the mutated lentiviral ENV protein.

The advantageous mutated Nef protein contains a substitution at position 93, as described in the international application WO 2006/018289 (Inventors: Renard M., Mangeney M. and Heidmann T.) and a deletion of the N-terminus of the protein involved in its myristoylation.

GAG expression will produce virus like particles (VLPs) which are particularly advantageous for a vaccine, in particular if the ENV protein is associated with the VLP (Guerbois et al., Virology, 388:191-203, 2009).

In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in the same vector as the one which also contains said at least one nucleic acid molecule coding for a GAG protein and/or a PRO protein and/or a POL protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.

In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in the same vector as the one which also contains all said at least one nucleic acid molecule coding for a GAG protein and/or a PRO protein and/or a POL protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.

In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in a vector which is different from the at least one vector containing said at least one nucleic acid molecule coding for a GAG protein and/or a PRO protein and/or a POL protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.

In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, said nucleic acid molecule coding for a GAG protein, said nucleic acid molecule coding for a PRO protein, said nucleic acid molecule coding for a POL protein and said nucleic acid molecule coding for a mutated NEF protein substantially devoid of immunosuppressive properties, are all contained in vectors which are different from each other.

In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, comprising at least one nucleic acid molecule coding for a GAG protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties,

-   -   of a human or simian lentivirus, said lentivirus being         preferably of the same origin as the mutated lentiviral ENV         protein.

In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in the same vector as the one which also contains said at least one nucleic acid molecule coding for a GAG protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.

In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in the same vector as the one which also contains a nucleic acid molecule coding for a GAG protein and for a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.

In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in the same vector as the one which also contains a nucleic acid molecule coding for a GAG protein and for a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties, said vector being preferably a measles vector (Guerbois et al., Virology, 388:191-203, 2009) or a canary pox vector (Poulet et al., Veterinary Record, 153(5):141-145, 2003; Vaccari et al., Expert Review of Vaccines, Vol. 9, No 9, pages 997-1005, 2010).

In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, is contained in a vector which is different from the at least one vector containing said at least one nucleic acid molecule coding for a GAG protein and/or a mutated NEF protein, wherein said mutated NEF protein is substantially devoid of immunosuppressive properties.

In another particular embodiment, the invention relates to a pharmaceutical composition, as defined above, wherein said nucleic acid molecule coding for a mutated human or simian lentiviral ENV protein, or a fragment of said mutated human or simian lentiviral ENV protein, said nucleic acid molecule coding for a GAG protein and said nucleic acid molecule coding for a mutated NEF protein substantially devoid of immunosuppressive properties, are all contained in vectors which are different from each other.

The invention also relates to a pharmaceutical composition according the above definition, for its use as vaccine, for the treatment or the prevention of lentiviral infection, in particular HIV-1 infection, HIV-2 infection and SIV infection.

As mentioned above the pharmaceutical composition according to the invention encompasses a vaccine, comprising as active substance, a protein as defined above, or a nucleic acid as defined above, or a vector as defined above, or a combination thereof.

In a particular embodiment, the pharmaceutical composition according to the invention encompasses a vaccine, comprising a nucleic acid coding for a GAG protein as defined above, a nucleic acid coding for a mutated ENV protein as defined above and a nucleic acid coding for a mutated NEF protein as defined above.

In a particular embodiment, the pharmaceutical composition according to the invention encompasses a vaccine as defined above, comprising a nucleic acid coding for a GAG protein as defined above, a nucleic acid coding for a mutated ENV protein as defined above and a nucleic acid coding for a mutated NEF protein as defined above, which are contained in a same vector, said same vector being preferably a canary pox vector or a measles vector, more preferably a measles vector.

In a particular embodiment, the pharmaceutical composition according to the invention encompasses a vaccine, comprising a GAG protein as defined above, a mutated ENV protein as defined above and a mutated NEF protein as defined above.

In a particular embodiment, the pharmaceutical composition according to the invention encompasses a vaccine, comprising a GAG protein as defined above, a mutated ENV protein as defined above and a mutated NEF protein as defined above which are associated to at least one adjuvant.

A comprehensive list of adjuvants that can be used in HIV vaccines is indicated in the FRANKLIN PIERCE LAW CENTER EDUCATIONAL REPORT: PATENT LANDSCAPE OF ADJUVANT FOR HIV VACCINES which is incorporated herein by reference. This list includes:

-   -   Aluminum-based compounds such as aluminum phosphate and aluminum         hydroxide.     -   Immunostimulatory adjuvants like CpG, MPL and QS21 orMF59 which         is a squalene oil-in water emulsions.     -   Freunds incomplete adjuvant (FIA) or Freund's complete adjuvant         (FCA), can also be used.     -   Calcium phosphate in particular orthophosphates, metaphosphates         or pyrophosphates and occasionally hydrogen or hydroxide ions.     -   Muramyl dipeptide (N-Acetyl muramyl-L-alanine-D-isoglutamine,         MDP) and its synthetic analogs such as muramyl tripeptide         phosphatidylethanolamine MTPPTdEtn),     -   Trehalose-6,6′-dimycolate,     -   Saponins (Triterpenoid glycosides) like QS-21     -   Stearyl Tyrosine (octadecyl ester hydrochloride salt of         tyrosine)     -   Immunostimulatory adjuvants can include the following classes of         compounds:     -   Polysaccharides like:         -   Chitosan:         -   Inulin:         -   Beta-Glucans         -   Lipo-Polysaccharides or endotoxin         -   MGN-3Actinidia eriantha (AEPS):         -   Eldexomer:         -   CpG ODN     -   Liposomes like         -   Dehydration-rehydration liposome vesicles (DRVs)         -   Cytotoxic T lymphocyte (CTL)         -   CAF01         -   Liposomes containing lipid A (LA)     -   Lipid Polysine Core Peptides (LCP)     -   Cytokine like GM-CSF, IL-2, IL-12 or IL-15     -   Lipid A and Monophosphoryl Lipid A (MPL)     -   Lipopeptide which are molecules consisting of lipid connected to         a peptide. They are derived from the lipoprotein of bacterial         cell wall.     -   Exogenous Immunostimulatory Adjuvants which are compounds, or         proteins that are not found in the human body like:         -   Proteosomes in particular Multiple Antigenic Peptides (MAP)             or Exogenous Toxins

Suitably, the total amount mutated lentiviral ENV protein in a single dose of the immunogenic composition is 10 μg and/or the total amount of unfused polypeptide in a single dose of the immunogenic composition is 20 μg. In one embodiment, the total amount of all antigens in a single dose of the immunogenic composition is 0.5-50 μg, 2-40 μg, 5-30 μg, 10μ-20 μg or around 30 μg, around 20 μg or around 10 μg.

The amount of mutated lentiviral ENV protein in a dose of the immunogenic composition is selected as an amount which induces an immune response without significant, adverse side effects in typical recipients. Such amount will vary depending upon which specific immunogen is employed and the dosing or vaccination regimen that is selected. An optimal amount for a particular immunogenic composition can be ascertained by standard studies involving observation of relevant immune responses in subjects.

Administration of the pharmaceutical composition can take the form of one or of more than one individual dose, for example as repeat doses of the same polypeptide containing composition, or in a heterologous “prime-boost” vaccination regime, including proteins and vectors. In one embodiment, the immunogenic composition of the invention is initially administered to a subject as two or three doses, wherein the doses are separated by a period of two weeks to three months, preferably one month.

Conveniently, the composition is administered to a subject (for instance as a booster) every 6-24, or 9-18 months, for instance annually. For instance, the composition is administered to a subject (for instance as a booster) at six month or 1 year intervals. Suitably in this respect, subsequent administrations of the composition to the subject boost the immune response of earlier administrations of the composition to the same subject.

In an embodiment, the immunogenic composition of the invention is used as part of a prime-boost regimen for use in the treatment or prevention of disease or infection by HIV-1 strains from one or more clades different from the one or more HIV-1 clades in the immunogenic composition. Conveniently, the composition is the priming dose. Alternatively, the composition is the boosting dose.

Suitably, two or more priming and/or boosting doses are administered. A heterologous prime-boost regime uses administration of different forms of immunogenic composition or vaccine in the prime and the boost, each of which can itself include two or more administrations. The priming composition and the boosting composition will have at least one antigen in common, although it is not necessarily an identical form of the antigen, it can be a different form of the same antigen.

Prime boost immunisations according to the invention can be homologous prime-boost regimes or heterologous prime-boost regimes. Homologous prime-boost regimes utilize the same composition for prime and boost, for instance the immunogenic composition of the invention. Heterologous prime-boost regimes can be performed with a combination of protein and DNA-based formulations. Such a strategy is considered to be effective in inducing broad immune responses. Adjuvanted protein vaccines induce mainly antibodies and CD4+ T cell immune responses, while delivery of DNA as a plasmid or a recombinant vector induces strong CD8+ T cell responses. Thus, the combination of protein and DNA vaccination can provide for a wide variety of immune responses. This is particularly relevant in the context of HIV, since neutralizing antibodies, CD4+ T cells and CD8+ T cells are thought to be important for the immune defense against HIV-1.

The invention also relates to a pharmaceutical composition according the above definition, for its use for the treatment of lentiviral infection.

The invention also relates to a method for treating patient afflicted by pathologies related to lentiviral infection, comprising the administration to a patient in a need thereof of a pharmaceutically efficient amount of the pharmaceutical composition as defined above. The invention also relates to a method for inducing an immune response against a lentivirus that has infected an individual, said method comprising the administration to a patient in a need thereof of a pharmaceutically efficient amount of the pharmaceutical composition as defined above.

The invention also relates to a pharmaceutical composition as defined above, further comprising an antiviral agent.

The composition according to the invention can also be used in combination with the antiviral compositions listed in Table 1 below:

TABLE 1 Trade names of the different antivirals used for HIV treatment, as well as their specificity of action. Intelence ® (TMC 125/etravirine) Tibotec - Non-nucleoside reverse transcriptase inhibitor Agenerase ® (APV/amprenavir) GSK - Protease inhibitor Aptivus ® (TPV/tipranavir) Boehringer - Protease inhibitor Crixivan ® (IDV/indinavir) MSD - Protease inhibitor Invirase ® (SQV/saquinavir) Roche - Protease inhibitor Kaletra ® (LPV.r/lopinavir + ritonavir) Protease inhibitor Abbott Norvir ® (ritonavir) Abbott - Protease inhibitor Prezista ® (TMC 114/darunavir) Protease inhibitor Tibotec/Janssen-Cilag - Reyataz ® (ATZ/atazanavir) BMS - Protease inhibitor Telzir ® (APV/fosamprenavir) GSK - Protease inhibitor Viracept ® (nelfinavir) Roche - Protease inhibitor Fuzeon ® (T20/enfuvirtide) Roche - Fusion inhibitor Celsentri ® (maraviroc) Pfizer - Entry inhibitor Isentress ® (MK 0518/raltegravir) Merck - Integrase inhibitor Rescriptor ® (delavirdine) Agouron - Non-nucleoside reverse transcriptase inhibitor Sustiva ® (EFV/efavirenz) BMS - Non-nucleoside reverse transcriptase inhibitor Viramune ® (nevirapine) Boehringer - Non-nucleoside reverse transcriptase inhibitor Combivir ® (Retrovir ® + Epivir-) GSK - Nucleoside reverse transcriptase inhibitor Emtriva ® (FTC, emtricitabine) Gilead - Nucleoside reverse transcriptase inhibitor Epivir ® (3TC, lamivudine) GSK - Nucleoside reverse transcriptase inhibitor Kivexa ® (Ziagen ® + Epivir ®) GSK - Nucleoside reverse transcriptase inhibitor Retrovir ®(AZT/zidovudine) QSK - Nucleoside reverse transcriptase inhibitor Trizivir ® (Retrovir ® + Epivir ® + Ziagen ®) Nucleoside reverse transcriptase inhibitor GSK - Videx ® (ddI/didanosine) BMS - Nucleoside reverse transcriptase inhibitor Viread ® (TDF/tenofovir) Gilead - Nucleoside reverse transcriptase inhibitor Zerit ® (d4T/stavudine) BMS - Nucleoside reverse transcriptase inhibitor Ziagen ® (ABC/abacavir) GSK - Nucleoside reverse transcriptase inhibitor Truvada ® (Emtriva ® + Viread ®) Gilead - Nucleoside reverse transcriptase inhibitor Atripla (Sustiva ® + Emtriva ® + Viread ®) Nucleoside and non-nucleoside reverse transcriptase BMS/GILEAD inhibitor

The invention also relates to a pharmaceutical composition as defined above, for simultaneous, separate or sequential use.

The invention relates to a pharmaceutical composition as defined above for its use for stimulating an immune response in a host organism.

The invention relates to a pharmaceutical composition as defined above for its use for inducing a specific immune response against the HIV ENV protein.

The invention relates to a pharmaceutical composition as defined above for its use for the prevention or the treatment of HIV infection, or pathologies related to AIDS.

The invention relates to a pharmaceutical composition as defined above, as a vaccine, for its use for inducing a specific immune response against the HIV ENV protein.

The invention relates to a pharmaceutical composition as defined above, as a vaccine, for its use for the prevention or the treatment of HIV infection, or pathologies related to AIDS.

In another aspect, the invention also relates to a method to obtain the active substance of a pharmaceutical composition, as defined above, consisting of modifying the immunosuppressive property of:

a wild-type human or simian lentiviral ENV protein,

or a fragment of said wild-type human or simian lentiviral ENV protein, said fragment comprising at least 40 amino acids,

said ENV protein or fragment thereof presenting a transmembrane subunit (TM) comprising an immunosuppressive domain (ISU) containing the following amino acid sequence:

(SEQ ID NO: 427) A-[I/V]-E-[K/R]-X′_(a)-X′_(b)-X-D-Q, wherein X represents any amino acid, X′_(a) is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and X′_(b) is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, said method comprising a step of introduction of at least one mutation of X′_(a) and/or X′_(b), to obtain: an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said mutated human or simian lentiviral ENV protein having at least 70% identity, preferably at least 80% identity, to one sequence chosen from the group consisting of SEQ ID NO: 216, SEQ ID NO: 420 and SEQ ID NO: 421, or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids, said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q, wherein X represents any amino acid, and either X_(a) is A, F, G, L, R or deleted, and X_(b) is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or X_(a) is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and X_(b) is A, F, G, R or deleted, or X_(a) is A, F, G, L, R or deleted, and X_(b) is A, F, G, R or deleted, said substantial absence of immunosuppressive activity of the above mentioned mutated human or simian lentiviral ENV protein or of the above defined fragment being liable to be assessed by the fact that in an in vivo assay involving engrafted tumor cells rejection, said tumor cells being transduced either so as to express said mutated ENV protein or said fragment (mutated ENV tumor cells), or said tumor cells being transduced so as to express said wild type ENV protein or a fragment thereof (wild type ENV tumor cells), or said tumor cells being not transduced (normal tumor cells), the following ratio: immunosuppression index of said mutated ENV protein or of said fragment (i_(mutated env))/immunosuppression index of wild type ENV protein (i_(wild type env)) is less than 0.5, i_(mutated env) being defined by: (maximum area reached by mutated ENV tumor cells−maximum area reached by normal tumor cells)/(maximum area reached by normal tumor cells), and i_(wild type env) being defined by: (maximum area reached by wild type ENV tumor cells−maximum area reached by normal tumor cells)/(maximum area reached by normal tumor cells).

The invention also relates to a method to obtain the active substance of a pharmaceutical composition, according to the above defined method, consisting of modifying the immunosuppressive property of:

a wild-type human or simian lentiviral ENV protein,

or a fragment of said wild-type human or simian lentiviral ENV protein, said fragment comprising at least 40 amino acids,

said ENV protein or fragment thereof presenting a transmembrane subunit (TM) comprising an immunosuppressive domain (ISU) containing the following amino acid sequence:

(SEQ ID NO: 1) A-[I/V]-E-[K/R]-Y-L-X-D-Q, wherein X represents any amino acid, said method comprising a step of introduction of at least one mutation of Y in position 5 and/or L in position 6, to obtain: an isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids, said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:

(SEQ ID NO: 416) A-[I/VN]-E-[K/R]-X_(a)-X_(b)-X-D-Q, wherein X represents any amino acid, and either X_(a) is A, F, G, L, R or deleted, and X_(b) is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or X_(a) is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and X_(b) is A, F, G, R or deleted, or X_(a) is A, F, G, L, R or deleted, and X_(b) is A, F, G, R or deleted.

The invention also relates to a method as defined above to obtain the active substance of a pharmaceutical composition,

wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,

or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,

said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q, wherein X represents any amino acid, and either X_(a) is A, F, G, L or R, and X_(b) is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or X_(a) is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and X_(b) is A, F, G or R, or X_(a) is A, F, G, L or R, and X_(b) is A, F, G or R.

The invention also relates to a method as defined above to obtain the active substance of a pharmaceutical composition,

wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,

or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,

said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q, wherein X represents any amino acid, and either X_(a) is A, F, G, L or R, and X_(b) is L, I, V, M or P, or X_(a) is Y, I, H, C or T, and X_(b) is A, F, G or R, or X_(a) is A, F, G, L or R, and X_(b) is A, F, G or R.

The invention also relates to a method as defined above to obtain the active substance of a pharmaceutical composition,

wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,

or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,

said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q, wherein X represents any amino acid, and either X_(a) is A, G, or R, and X_(b) is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or X_(a) is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and X_(b) is F, G or R, or X_(a) is F or L, and X_(b) is F, G or R, or X_(a) is A, G or R, and X_(b) is A.

The invention also relates to a method as defined above to obtain the active substance of a pharmaceutical composition,

wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,

or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,

said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q, wherein X represents any amino acid, and either X_(a) is A, G, or R, and X_(b) is L, I, V, M or P, or X_(a) is Y, I, H, C or T, and X_(b) is F, G or R, or X_(a) is F or L, and X_(b) is F, G or R, or X_(a) is A, G or R, and X_(b) is A, or X_(a) is A, G or R, and X_(b) is F, G or R.

The invention also relates to a method as defined above to obtain the active substance of a pharmaceutical composition,

wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,

or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,

said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q, wherein, X represents any amino acid, and either X_(a) is A, G, or R, and X_(b) is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or X_(a) is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and X_(b) is F, G or R.

The invention also relates to a method as defined above to obtain the active substance of a pharmaceutical composition,

wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,

or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,

said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q, wherein, X represents any amino acid, and either X_(a) is A, G, or R, and X_(b) is L, I, V, M or P, or X_(a) is Y, I, H, C or T, and X_(b) is F, G or R.

The invention also relates to a method as defined above to obtain the active substance of a pharmaceutical composition,

wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,

or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,

said mutated ENV protein and fragment thereof comprising a mutated immunosuppressive domain (ISU) containing the following amino sequence:

(SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q, wherein, X represents any amino acid, and either X_(a) is R, and X_(b) is C, D, E, H, I, K, L, M, N, P, Q, S, T, V, W or Y, or X_(a) is C, D, E, H, I, K, M, N, P, Q, S, T, V, W or Y, and X_(b) is R, or X_(a) is R, and X_(b) is R.

The invention also relates to a method as defined above to obtain the active substance of a pharmaceutical composition,

wherein said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity,

or a fragment of said isolated mutated human or simian lentiviral ENV protein having substantially no immunosuppressive activity, said fragment comprising at least 40 amino acids,

said mutated ENV protein and fragment thereof comprising a mutation in one at least of the amino acids at positions, 29, 36 and 37 of SEQ ID NO: 426:

(SEQ ID NO: 426) A[I/V]E[K/R]X_(a)X_(b)X₁DQX₂X₃LX₄X₅WGC[A/S][F/G]X₆X₇CVX₈ TX₉VPX_(c)X₁₀Z₁Z₂Z₃Z₄Z₅X_(d)X_(e)[S/T] wherein X_(a) and X_(b) are as defined in anyone of claims 1 to 15, X₁ to X₁₀ represent any amino acid, Z₁ to Z₅ represent no amino acid or any amino acid, independently from each other such that

-   -   X_(c) is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,         Y, or deleted, preferably A, D, or N,     -   X_(d) is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, Y,         W, or deleted, preferably A, G, S or Y,     -   X_(e) is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, Y,         W, or deleted, preferably A, D or N.

The present invention relates to a pharmaceutical composition comprising as active substance:

a) an isolated non naturally occurring mutated human or simian lentiviral ENV having substantially no immunosuppressive activity, said mutated human or simian lentiviral ENV resulting from mutation of the transmembrane subunit (TM) of a wild type human or simian lentiviral ENV protein, the transmembrane subunit comprising an immunosuppressive domain (ISU) comprising the following amino acid sequence:

(SEQ ID NO: 1) A-[I/V]-E-[K/R]-Y-L-X-D-Q, said sequence encompassing

-   -   AIEKYLXDQ (SEQ ID NO: 2), AIERYLXDQ (SEQ ID NO: 3), AVEKYLXDQ         (SEQ ID NO: 4) and AVERYLXDQ (SEQ ID NO: 5), wherein X         represents any natural amino acid,         wherein the amino acids at the positions chosen among     -   the position 4 of SEQ ID NO: 1,     -   the position 5 of SEQ ID NO: 1,     -   the position 6 of SEQ ID NO: 1,     -   the positions 4 and 5 of SEQ ID NO: 1,     -   the position 4 and 6 of SEQ ID NO: 1,     -   the position 5 and 6 of SEQ ID NO: 1, and     -   the position 4, 5 and 6 of SEQ ID NO: 1,         are:     -   either deleted,     -   or substituted by another amino acid such that         -   when the immunosuppressive domain comprises SEQ ID NO: 2 or             4             -   the amino acid residue at position 4 of SEQ ID NO: 1 is                 substituted by A, C, D, E, F, G, H, I, L, M, N, P, Q, R,                 S, T, V, W or Y, and/or             -   the amino acid residue at position 5 of SEQ ID NO: 1 is                 substituted by A, C, D, E, F, G, I, L, M, N, P, Q, R, S,                 T, V or W, and/or             -   the amino acid residue at position 6 of SEQ ID NO: 1 is                 substituted by A, C, D, E, F, G, H, I, K, M, N, P, Q, R,                 S, T, V, W or Y,         -   when the immunosuppressive domain comprises SEQ ID NO: 3 or             5,             -   the amino acid residue at position 4 of SEQ ID NO: 1 is                 substituted by A, C, D, E, F, G, H, I, K, L, M, N, P, Q,                 S, T, V, W or Y, and/or             -   the amino acid residue at position 5 of SEQ ID NO: 1 is                 substituted by A, C, D, E, F, G, I, L, M, N, P, Q, R, S,                 T, V or W, and/or             -   the amino acid residue at position 6 of SEQ ID NO: 1 is                 substituted by A, C, D, E, F, G, H, I, K, M, N, P, Q, R,                 S, T, V, W or Y,                 or                 b) a fragment of said mutated human or simian lentiviral                 ENV, said fragment comprising at least the                 immunosuppressive domain (ISU) of said wild type human                 or simian lentiviral ENV comprising the following amino                 acid sequence:

(SEQ ID NO: 1) A-[I/V]-E-[K/R]-Y-L-X-D-Q, wherein the amino acids at the positions chosen among

-   -   the position 4 of SEQ ID NO: 1,     -   the position 5 of SEQ ID NO: 1,     -   the position 6 of SEQ ID NO: 1,     -   the positions 4 and 5 of SEQ ID NO: 1,     -   the position 4 and 6 of SEQ ID NO: 1,     -   the position 5 and 6 of SEQ ID NO: 1, and     -   the position 4, 5 and 6 of SEQ ID NO: 1,         are:     -   either deleted,     -   or substituted by another amino acid such that         -   when the immunosuppressive domain comprises SEQ ID NO: 2 or             4             -   the amino acid residue at position 4 of SEQ ID NO: 1 is                 substituted by A, C, D, E, F, G, H, I, L, M, N, P, Q, R,                 S, T, V, W or Y, and/or             -   the amino acid residue at position 5 of SEQ ID NO: 1 is                 substituted by A, C, D, E, F, G, I, L, M, N, P, Q, R, S,                 T, V or W, and/or             -   the amino acid residue at position 6 of SEQ ID NO: 1 is                 substituted by A, C, D, E, F, G, H, I, K, M, N, P, Q, R,                 S, T, V, W or Y,         -   when the immunosuppressive domain comprises SEQ ID NO: 3 or             5,             -   the amino acid residue at position 4 of SEQ ID NO: 1 is                 substituted by A, C, D, E, F, G, H, I, K, L, M, N, P, Q,                 S, T, V, W or Y, and/or             -   the amino acid residue at position 5 of SEQ ID NO: 1 is                 substituted by A, C, D, E, F, G, I, L, M, N, P, Q, R, S,                 T, V or W, and/or             -   the amino acid residue at position 6 of SEQ ID NO: 1 is                 substituted by A, C, D, E, F, G, H, I, K, M, N, P, Q, R,                 S, T, V, W or Y,                 said fragment having substantially no immunosuppressive                 properties,                 in association of a pharmaceutically acceptable carrier.

In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein the amino acids at the positions of the group consisting of

-   -   the position 4 of SEQ ID NO: 1,     -   the position 5 of SEQ ID NO: 1,     -   the position 6 of SEQ ID NO: 1,     -   the positions 4 and 5 of SEQ ID NO: 1,     -   the position 4 and 6 of SEQ ID NO: 1,     -   the position 5 and 6 of SEQ ID NO: 1, and     -   the position 4, 5 and 6 of SEQ ID NO: 1,         -   are substituted by another amino acid such that             -   when the immunosuppressive domain comprises SEQ ID NO: 2                 or 4                 -   the amino acid residue at position 4 of SEQ ID NO: 1                     is substituted by A, C, D, E, F, G, H, I, L, M, N,                     P, Q, R, S, T, V, W or Y, and/or                 -   the amino acid residue at position 5 of SEQ ID NO: 1                     is substituted by A, C, D, E, F, G, I, L, M, N, P,                     Q, R, S, T, V or W, and/or                 -   the amino acid residue at position 6 of SEQ ID NO: 1                     is substituted by A, C, D, E, F, G, H, I, K, M, N,                     P, Q, R, S, T, V, W or Y,             -   when the immunosuppressive domain comprises SEQ ID NO: 3                 or 5,                 -   the amino acid residue at position 4 of SEQ ID NO: 1                     is substituted by A, C, D, E, F, G, H, I, K, L, M,                     N, P, Q, S, T, V, W or Y, and/or                 -   the amino acid residue at position 5 of SEQ ID NO: 1                     is substituted by A, C, D, E, F, G, I, L, M, N, P,                     Q, R, S, T, V or W, and/or                 -   the amino acid residue at position 6 of SEQ ID NO: 1                     is substituted by A, C, D, E, F, G, H, I, K, M, N,                     P, Q, R, S, T, V, W or Y

Advantageously, the mutated lentiviral protein according to the invention, comprised in the above defined composition, comprises the amino acid sequence of the list consisting of SEQ ID NO: 407 to SEQ ID NO: 414.

The correspondence is the following one:

SEQ ID NO: 407 AVEAALKD SEQ ID NO: 408 AVEEALKD SEQ ID NO: 409 AVESALKD SEQ ID NO: 410 AVETALKD SEQ ID NO: 411 AIEAALKD SEQ ID NO: 412 AIEEALKD SEQ ID NO: 413 AIESALKD SEQ ID NO: 414 AIETALKD

These proteins have been mutated at position 4 of SEQ ID NO: 1, by substitution, and the amino acid at position 5 has been substituted by A.

In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein the amino acids at the positions of the group consisting of

-   -   the position 5 of SEQ ID NO: 1,     -   the position 6 of SEQ ID NO: 1, and     -   the position 5 and 6 of SEQ ID NO: 1,         -   are substituted by another amino acid such that             -   the amino acid residue at position 5 of SEQ ID NO: 1 is                 substituted by A, C, D, E, F, G, I, L, M, N, P, Q, R, S,                 T, V or W, and/or             -   the amino acid residue at position 6 of SEQ ID NO: 1 is                 substituted by A, C, D, E, F, G, H, I, K, M, N, P, Q, R,                 S, T, V, W or Y.

In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said immunosuppressive domain (ISU) comprises the amino acid sequence of the group consisting of: SEQ ID NO: 4 or 5.

In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said immunosuppressive domain (ISU) comprises the amino acid sequence SEQ ID NO: 5, and wherein

-   -   the amino acid residue at position 5 of SEQ ID NO: 5 is         substituted by A, F, G, L or R, or     -   the amino acid residue at position 6 of SEQ ID NO: 5 is         substituted by A, F, G or R, or     -   the amino acid residues at position 5 and 6 of SEQ ID NO: 5 are         substituted by A, F, G, or R.

This is the case when said ENV protein is the ENV protein of HIV 1 virus.

The invention also relates to a pharmaceutical composition as defined above, wherein said immunosuppressive domain (ISU) comprises the amino acid sequence SEQ ID NO: 2, and wherein

-   -   the amino acid residue at position 5 of SEQ ID NO: 2 is         substituted by A, F, G, L or R, or     -   the amino acid residue at position 6 of SEQ ID NO: 2 is         substituted by A, F, G or R, or     -   the amino acid residues at position 5 and 6 of SEQ ID NO: 2 are         substituted by A, F, G, or R.

The invention also relates to a pharmaceutical composition as defined above, wherein said immunosuppressive domain (ISU) comprises the amino acid sequence SEQ ID NO: 3, and wherein

-   -   the amino acid residue at position 5 of SEQ ID NO: 3 is         substituted by A, F, G, L or R, or     -   the amino acid residue at position 6 of SEQ ID NO: 3 is         substituted by A, F, G or R, or     -   the amino acid residues at position 5 and 6 of SEQ ID NO: 3 are         substituted by A, F, G, or R.

In one advantageous embodiment, the invention relates to a pharmaceutical composition as defined above, wherein said mutated protein further comprises the wild type amino acid sequence SEQ ID NO: 270,

(SEQ ID NO: 270) X₁DQX₂X₃LX₄X₅WGC[A/S][F/G]X₆X₇CVX₈TX₉VPWX₁₀Z₁Z₂Z₃ Z₄Z₅[N/S][E/D/N/A][S/T] wherein X₁-X₁₀ represents any amino acid, wherein Z₁ to Z₅ represent no amino acid or any natural amino acid, independently from each other, said mutated lentiviral protein further comprising a mutation in one at least of the amino acids at positions 23, 30 and 31 of SEQ ID NO: 270

-   -   said mutation being:         -   either a deletion,         -   or a substitution such that             -   the amino acid at position 25 of SEQ ID NO: 270 is                 substituted by A, C, D, E, F, G, H, I, K, L, M, N, P, Q,                 R, S, T, V, Y,             -   the amino acid at position 32 of SEQ ID NO: 270 is                 substituted by A, C, D, E, F, G, H, I, K, L, M, P, Q, R,                 T, V, Y, W,             -   the amino acid at position 33 of SEQ ID NO: 270 is                 substituted by C, F, G, H, I, K, L, M, P, Q, R, S, T, V,                 Y, W.

In one advantageous embodiment, the invention relates to a pharmaceutical composition comprising as active substance:

an isolated non naturally occurring mutated human or simian lentiviral ENV having substantially no immunosuppressive activity, said mutated human or simian lentiviral ENV resulting from mutation of a wild type human or simian lentiviral ENV protein, said wild type human or simian lentiviral ENV protein comprising the following amino acid sequence:

(SEQ ID NO: 415) A[I/V]E[K/R]YLX₁DQX₂X₃LX₄X₅WGC[A/S][F/G]X₆X₇CVX₈ TX₉VPWX₁₀Z₁Z₂Z₃Z₄Z₅[N/S][E/D/N/A][S/T], wherein X₁-X₁₀ represents any amino acid, wherein Z₁ to Z₅ represent no amino acid or any natural amino acid, independently from each other, wherein the amino acids at the positions chosen among

-   -   the position 4 of SEQ ID NO: 1,     -   the position 5 of SEQ ID NO: 1,     -   the position 6 of SEQ ID NO: 1,     -   the positions 4 and 5 of SEQ ID NO: 1,     -   the position 4 and 6 of SEQ ID NO: 1,     -   the position 5 and 6 of SEQ ID NO: 1, and     -   the position 4, 5 and 6 of SEQ ID NO: 1,         are:     -   either deleted,     -   or substituted by another amino acid such that         -   the amino acid residue at position 4 of SEQ ID NO: 1 is             substituted by A, C, D, E, F, G, H, I, L, M, N, P, Q, S, T,             V, W or Y, and/or         -   the amino acid residue at position 5 of SEQ ID NO: 1 is             substituted by A, C, D, E, F, G, I, L, M, N, P, Q, R, S, T,             V or W, and/or         -   the amino acid residue at position 6 of SEQ ID NO: 1 is             substituted by A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,             T, V, W or Y,             and further wherein             one at least of the amino acids at positions 29, 36 and 37             of the amino acid sequence SEQ ID NO: 415 are mutated, by     -   either a deletion,     -   or a substitution such that         -   the amino acid at position 29 of SEQ ID NO: 415 is             substituted by A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,             S, T, V, Y,         -   the amino acid at position 36 of SEQ ID NO: 415 is             substituted by A, C, D, E, F, G, H, I, K, L, M, P, Q, R, T,             V, Y, W,         -   the amino acid at position 37 of SEQ ID NO: 415 is             substituted by C, F, G, H, I, K, L, M, P, Q, R, S, T, V, Y,             W.

In another aspect, the invention relates to a pharmaceutical composition as defined above, further comprising a mutation at the position X₄

(SEQ ID NO: 270) X₁DQX₂X₃LX₄X₅WGC[A/S][F/G]X₆X₇CVX₈TX₉VPWX₁₀Z₁Z₂Z₃ Z₄Z₅[N/S][E/D/N/A][S/T] wherein X₁-X₃ and X₅-X₁₀ represents any amino acid,

-   Z₁ to Z₅ represent no amino acid or any amino acid, independently     from each other said mutation being:     -   either a deletion,     -   or a substitution by A, C, D, E, F, H, I, K, L, M, P, Q, R, S,         T, V, Y or W.

In one advantageous embodiment, the invention relates to a pharmaceutical composition comprising as active substance:

an isolated non naturally occurring mutated human or simian lentiviral ENV having substantially no immunosuppressive activity, said mutated human or simian lentiviral ENV resulting from mutation of a wild type human or simian lentiviral ENV protein, said wild type human or simian lentiviral ENV protein comprising the following amino acid sequence:

(SEQ ID NO: 415) A[I/V]E[K/R]YLX₁DQX₂X₃LX₄X₅WGC[A/S][F/G]X₆X₇CVX₈ TX₉VPWX₁₀Z₁Z₂Z₃Z₄Z₅[N/S][E/D/N/A][S/T], wherein X₁-X₁₀ represents any amino acid, wherein Z₁ to Z₅ represent no amino acid or any natural amino acid, independently from each other, wherein the amino acids at the positions chosen among

-   -   the position 4 of SEQ ID NO: 1,     -   the position 5 of SEQ ID NO: 1,     -   the position 6 of SEQ ID NO: 1,     -   the positions 4 and 5 of SEQ ID NO: 1,     -   the position 4 and 6 of SEQ ID NO: 1,     -   the position 5 and 6 of SEQ ID NO: 1, and     -   the position 4, 5 and 6 of SEQ ID NO: 1,         are:     -   either deleted,     -   or substituted by another amino acid such that         -   the amino acid residue at position 4 of SEQ ID NO: 1 is             substituted by A, C, D, E, F, G, H, I, L, M, N, P, Q, S, T,             V, W or Y, and/or         -   the amino acid residue at position 5 of SEQ ID NO: 1 is             substituted by A, C, D, E, F, G, I, L, M, N, P, Q, R, S, T,             V or W, and/or         -   the amino acid residue at position 6 of SEQ ID NO: 1 is             substituted by A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,             T, V, W or Y,             and possibly, further wherein             one at least of the amino acids at positions 29, 36 and 37             of the amino acid sequence SEQ ID NO: 415 are mutated, by     -   either a deletion,     -   or a substitution such that         -   the amino acid at position 29 of SEQ ID NO: 415 is             substituted by A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,             S, T, V, Y,         -   the amino acid at position 36 of SEQ ID NO: 415 is             substituted by A, C, D, E, F, G, H, I, K, L, M, P, Q, R, T,             V, Y, W,         -   the amino acid at position 37 of SEQ ID NO: 415 is             substituted by C, F, G, H, I, K, L, M, P, Q, R, S, T, V, Y,             W,             and/or possibly further wherein the amino acid at position             X₄ is either deleted or substituted by A, C, D, E, F, H, I,             K, L, M, P, Q, R, S, T, V, Y or W, preferably by R or H.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1:

Amino acid sequences of the HIV ectodomain deletants, with the indicated length, and of single mutant (substitution are underlined) ectodomains.

FIG. 2:

Functional delineation of the immunosuppressive domain of the HIV envelope. The immunosuppressive activity of 115aa-long and truncated HIV envelope ectodomains (see structures on the left) was tested using the MCA205 tumor rejection in vivo assay. Immunosuppression indexes are given as histograms on the right (mean values+/−SD).

FIG. 3:

Functional identification of the aminoacid in the HIV envelope ectodomain directly involved in immunosuppressive activity, and search for aminoacid substitutions inhibiting this activity. Immunosuppressive activity was tested in vivo as in FIG. 2.

FIG. 4:

Expression profile of mutated HIV envelopes using full-length HIVenv-expressing vectors and an anti-SU HIVenv(110H) monoclonal antibody (FACS). The data are expressed as percentages of the control HIVenv WT.

FIG. 5:

Infectivity of HIVenv-pseudotyped viral particles showing functionality of the full-length WT and of specifically mutated HIV envelopes. The results are expressed as viral titers (number of infected target cells/mL of virus supernatant).

FIG. 6:

Functional identification of the amino acid in the SIV envelope ectodomain directly involved in immunosuppressive activity, and search for aminoacid substitutions inhibiting this activity. Immunosuppressive activity was tested using the in vivo MCA205 tumor rejection assay. Immunosuppression indexes are represented as histograms (mean values+/−SD).

DETAILED DESCRIPTION OF THE INVENTION Example

Immunosuppressive domains have been identified on the envelope proteins of oncoretroviruses, of either the gamma-(murine MLV) or the delta (e.g. human HTLV) type, as well as of some endogenous retroviruses (e.g. HERV-FRD). These domains have a highly conserved crystallographic structure, although their primary sequences are quite diverse, and an amino-acid (either Q, E, or K) at a definite position has previously been demonstrated to be essential for the immunosuppressive activity of the corresponding envelope protein. Mutation of this amino acid to an Arginine (R) was further demonstrated to result in inhibition of the immunosuppressive activity of the mutated envelope protein, with in some cases complete conservation of the other functional properties of the envelope protein, including its ability to be normally expressed at the cell membrane, to be captured by a nascent retroviral particle, and finally to confer infectivity of the mutant virus in vitro. Such mutants allowed an unambiguous demonstration of the essential role of the immunosuppressive activity for viremia in vivo, with the mutant IS-virus being unable to escape the host immune system and propagate in an immunocompetent animal [Schlecht-Louf et al., Proc Natl Acad Sci USA. 2010; 107(8):3782-7].

On the basis of the identification of an IS domain and on the ability of the IS function to be inhibited by specific mutations within the IS domain that do not disrupt the antigenic structure of the corresponding viral protein, vaccinal approaches are being developed with vaccines containing mutated <<optimized>> IS-negative viral antigens, which demonstrate an increased immunogenicity as compared to those using the native viral antigens. Search for similar IS domains and appropriate mutations in the case of non-oncogenic—but still pathogenic-retroviruses such as HIV is therefore of interest to tentatively develop improved vaccines.

Actually, in the case of the other major class of retroviruses, namely the lentiviruses (among which the human HIV1 and HIV2, and the SIV simian homologues), the crystallographic structure of the envelope protein discloses some similarities but also very important differences, especially in the domain corresponding to the IS domain of oncoretroviruses, with evidence in HIV and SIV for a severely extended helix-loop-helix domain, and no evidence for sequence similarities with the IS domain of oncoretroviruses. Furthermore, an IS domain with strong amino-acid similarities with the IS domain of oncoretroviruses was identified within an accessory protein of HIV and SIV, namely within the Nef protein. This protein is specifically produced by these complex retroviruses and is not encoded by oncoretroviruses. The Nef protein is essential for viremia in vivo, and it possesses several domains responsible for immune escape, among which the identified IS domain, thus strongly suggesting that lentiviruses had transferred the immunosuppressive activity found in oncoretroviruses within their envelope protein, to the accessory Nef protein. In agreement with this hypothesis, Nef-deleted or Nef-mutated retroviruses have a severely attenuated pathogenicity in in vivo macaque animal models. Here, the Inventors report on the identification of an IS activity carried by the HIV and SIV envelope proteins, and on specific mutations that inhibit this activity without major disrupt ion of the overall structure of the corresponding envelope proteins.

Results

Delineation of the Immunosuppressive Domain of the HIV Envelope

Delineation of the immunosuppressive domain of the HIV envelope was achieved using an in vivo tumor rejection assay, that the Inventors had previously used to demonstrate the immunosuppressive activity of the Env protein of oncoretroviruses (murine MoMLV and simian MPMV). The rationale of the assay can be summarized as follows: while injection of MCA205 tumor cells (H-2^(b)) into allogeneic Balb/c mice (H-2^(d)) leads to the formation of no tumor or transient tumors that are rapidly rejected, injection of the same cells, but stably expressing an immunosuppressive retroviral Env protein, leads to the growth of larger tumors that persist for a longer time—in spite of the expression of the new exogenous antigen. This difference is not associated with a difference in intrinsic cell growth rate since it is not observed in syngeneic C57BL/6 mice, and is immune system-dependent. The extent of “immunosuppression” can be quantified by an index based on tumor size: (A_(env)−A_(none))/A_(none), where A_(env) and A_(none) are the mean areas at the peak of growth of tumors from Balb/c mice injected with env-expressing or control cells, respectively. A positive index indicates that env expression facilitates tumor growth, as a consequence of its immunosuppressive activity; a null or negative index points to no effect or even an inhibitory effect, respectively. The latter may be explained by a stimulation of the immune response of the host against the new foreign antigen, represented by a non-immunosuppressive Env protein, expressed at the surface of tumor cells.

To first delineate a minimal immunosuppressive domain (ISD) active in vivo, the Inventors analyzed the effect of a series of truncations/deletions within the HIV env gene. Assay of the series of C-term truncations in FIGS. 1&2 identified a 49 residue-long domain with IS activity, with the 6 aa shorter HIV43 fragment and subsequent C-term truncated HIV37 and HIV30 fragments being IS-negative. HIV49 is embedded into the so-called ectodomain, which corresponds to a soluble part of the extracellular domain of the TM subunit, and consists in the a-helical domain involved in HIV TM trimerization, and the N-term part of the loop containing the 2 well-conserved, 6 aa-distant, cysteine residues found in most retroviral envelopes. Refine delineation of the amino acid responsible for IS activity was then performed by arginine-scanning between aa 36 and 51, i.e. in the domain associated with the transition from IS positive to negative Env subdomains. As illustrated in FIG. 3, all the X-to-Arg substitutions resulted in no significant change in the IS activity of HIV115, with one exception for Y41 and L42, which resulted in loss of IS activity of the mutant peptide.

Mutations at Position 41

The Inventors then checked that the Y41R substitution did not alter the overall capacity of the HIV envelope to be expressed by an eucaryotic cell and to be exported at the cell membrane, by introducing the Y41R substitution into an expression vector for the HIV envelope. A FACS analysis of cell transfected with both the wild-type and the Y41R mutant using an anti-SU specific monoclonal antibody actually demonstrated quantitative expression of the mutant envelope at the cell surface, thus indicating that the Y41R mutation does not significantly alter the HIV Env structure and SU-TM interaction (FIG. 4).

A series of distinct substitutions at the Y41 position were finally performed to tentatively identify whether other amino acids could be substituted to generate IS-negative variants: the amino acid assayed included the positively charged K and H (in addition to R), the small A and G, and the hydrophobic L and F residues. Again it was checked that these substitutions did not alter the overall structure of the HIV Env with the corresponding mutations.

Among them, substitution of Y41 by A, G, L and F resulted in loss of IS (FIG. 3), and did not alter the overall structure of the HIV Env with the corresponding mutations (FIGS. 4 and 5).

Further Mutation Outside of the ISU Domain

The effects of a set of second mutations at positions that are structurally close to Y41 position within the ENV ectodomain three-dimensional structure were also tested. Two of them (Y41R-K72A and Y41R-K78G) maintain a high level expression at cell membrane and confer infectivity (FIG. 5).

Mutations at Position 42

Interestingly, the adjacent position to the position Y41, position L42, also resulted in loss of IS activity of the mutant peptide when the mutation L42R is introduced. This mutation maintains a high level expression at cell membrane of the mutated HIV ENV protein (FIG. 4).

Mutations within the SIV ENV Protein

Interestingly, mutation at the homologous position in SIV ENV (L42) of the position L42 in HIV-1 ENV ectodomain also resulted in specific loss of IS activities (FIG. 6).

Accordingly, the present investigation has clearly identified a definite location and a definite substitution(s) within the HIV env resulting in the loss of its IS activity. Being compatible with the conservation of the overall structure of the human and simian lentiviral Env proteins, these substitutions should be introduced in all pharmaceutical preparations which include the Env protein as a vaccine antigen.

Materials and Methods

Mice and Cell Lines:

C57Bl/6 and Balb/c mice, 6-10 weeks old, were obtained from CER Janvier (Laval, France). Mice were maintained in the animal facility of the Gustave Roussy Institute in accordance with institutional regulations. 293T (ATCC CRL11268), and MCA205 cells were cultured in DMEM supplemented with 10% fetal calf serum (Invitrogen), streptomycin (100 μg/ml) and penicillin (100 units/ml).

Plasmid Construction:

The PCEL/E160 encoding the envelope protein of the BRU/LAI HIV-1 isolate is a gift from Dr Marc Sitbon. To generate the pDFG plasmids encoding the various fragments of HIV-1 envelope ectodomain, PCR fragments generated using PCEL/E160 as a template and primer pairs 1-2 (pDFG-HIV115), 1-3 (pDFG-HIV81), 1-4 (pDFG-HIV67), 1-5 (pDFG-HIV55), 1-6 (pDFG-HIV49), 1-7 (pDFG-HIV43), 1-8 (pDFG-HIV37), 1-9 (pDFG-HIV30) were digested with SfiI and MluI and inserted into pDFG-ectoSyncytin-1 (Mangeney et al, 2007) opened with the same enzymes.

Mutated pDFG-HIV115 were obtained by successive PCR using appropriate primers. A first series of PCR was performed with pDFG HIV115 as template using primers 1-11 and 10-2, 1-13 and 12-2, 1-15 and 14-2 or 1-17 and 16-2 to introduce the E39R, Y41R, K43R or D44R mutations respectively, and primers 1-18 and 19-2, 1-20 and 21-2, 1-22 and 23-2, 1-24 and 25-2, 1-26 and 27-2, 1-28 and 29-2, 1-30 and 31-2, 1-32 and 33-2, 1-34 and 35-2, or 1-36 and 37-2 to introduce the L36R, A37R, V38R, L42R, Q45R, Q46R, L47R, L48R, G49R, or 150R mutations respectively. The PCR products were then used as templates in subsequent PCR using primers 1-2. These PCR fragments were digested with SfiI and MluI and inserted into pDFG-ectoSyncytin-1 (Mangeney et al, 2007) opened with the same enzymes.

All the constructions were sequenced before use.

TABLE 1 Primer list N^(o) Name Primer sequence (5′-3′) SEQ ID 1 TMHIV Sfi-Sens ACATggcccagccggccTCTGGTATAGTGCAGCAGC SEQ ID NO: 295 2 TMHIV115 Mlu-AS GTATacgcgtTTATAATTCTTGTTCATTCTTTTC SEQ ID NO: 296 3 TMHIV81 Mlu AS GTATacgcgtTTACATGTTATTCCAAATCTGTTCC SEQ ID NO: 297 4 TMHIV67 Mlu AS GTATacgcgtTTAAGCATTCCAAGGCACAGC SEQ ID NO: 298 5 TMHIV55 Mlu AS GTATacgcgtTTATCCAGAGCAACCCCAAATCC SEQ ID NO: 299 6 TMHIV49 Mlu AS GTATacgcgtTTACCCCAGGAGCTGTTGATCC SEQ ID NO: 300 7 TMHIV43 Mlu AS GTATacgcgtTTACTTTAGGTATCTTTCCACAGC SEQ ID NO: 301 8 TMHIV37 Mlu AS GTATacgcgtTTAAGCCAGGATTCTTGCCTGGAG SEQ ID NO: 302 9 TMHIV30 Mlu AS GTATacgcgtTTACTGCTTGATGCCCCAGAC SEQ ID NO: 303 18 TMHIV115 L36R as CTTTCCACAGCCcGGATTCTTGCCTG SEQ ID NO: 304 19 TMHIV115 L36R s CAGGCAAGAATCCgGGCTGTGGAAAG SEQ ID NO: 305 20 TMHIV115 A37R as GTATCTTTCCACAcgCAGGATTCTTGCC SEQ ID NO: 306 21 TMHIV115 A37R s GGCAAGAATCCTGcgTGTGGAAAGATAC SEQ ID NO: 307 22 TMHIV115 V38R as CTTTAGGTATCTTTCCctAGCCAGGATTCTTGCC SEQ ID NO: 308 23 TMHIV115 V38R s GGCAAGAATCCTGGCTagGGAAAGATACCTAAAG SEQ ID NO: 309 11 TMHIV115 E39R AS CCTTTAGGTATCTTctCACAGCCAGGATTC SEQ ID NO: 310 10 TMHIV115 E39R S GAATCCTGGCTGTGagAAGATACCTAAAGG SEQ ID NO: 311 13 TMHIV115 Y41R AS GTTGATCCTTTAGGcgTCTTTCCACAGCCAG SEQ ID NO: 312 12 TMHIV115 Y41R S CTGGCTGTGGAAAGAcgCCTAAAGGATCAAC SEQ ID NO: 313 24 TMHIV115 L42R as GGAGCTGTTGATCCTTTcGGTATCTTTCCACAGCC SEQ ID NO: 314 25 TMHIV115 L42R s GGCTGTGGAAAGATACCgAAAGGATCAACAGCTCC SEQ ID NO: 315 15 TMHIV115 K43R AS GAGCTGTTGATCCcTTAGGTATCTTTCCAC SEQ ID NO: 316 14 TMHIV115 K43R S GTGGAAAGATACCTAAgGGATCAACAGCTC SEQ ID NO: 317 17 TMHIV115 D44R AS AGGAGCTGTTGAcgCTTTAGGTATCTTT SEQ ID NO: 318 16 TMHIV115 D44R S AAAGATACCTAAAGcgTCAACAGCTCCT SEQ ID NO: 319 26 TMHIV115 Q45R as CCCAGGAGCTGTcGATCCTTTAGGTATC SEQ ID NO: 320 27 TMHIV115 Q45R s GATACCTAAAGGATCgACAGCTCCTGGG SEQ ID NO: 321 28 TMHIV115 Q46R as CAAATCCCCAGGAGCcGTTGATCCTTTAG SEQ ID NO: 322 29 TMHIV115 Q46R s CTAAAGGATCAACgGCTCCTGGGGATTTG SEQ ID NO: 323 30 TMHIV115 L47R as CAAATCCCCAGGcGCTGTTGATCCTTTAG SEQ ID NO: 324 31 TMHIV115 L47R s CTAAAGGATCAACAGCgCCTGGGGATTTG SEQ ID NO: 325 32 TMHIV115 L48R as CCCAAATCCCCcGGAGCTGTTG SEQ ID NO: 326 33 TMHIV115 L48R s CAACAGCTCCgGGGGATTTGGG SEQ ID NO: 327 34 TMHIV115 G49R as CCCCAAATCCgCAGGAGCTGTTG SEQ ID NO: 328 35 TMHIV115 G49R s CAACAGCTCCTGcGGATTTGGGG SEQ ID NO: 329 36 TMHIV115 150R as GCAACCCCAtcTCCCCAGGAGCTG SEQ ID NO: 330 37 TMHIV115 150R s CAGCTCCTGGGGAgaTGGGGTTGC SEQ ID NO: 331 38 TMHIV115 W51R as GCAACCCCAtcTCCCCAGGAGCTG SEQ ID NO: 332 39 TMHIV115 W51R s CAGCTCCTGGGGAgaTGGGGTTGC SEQ ID NO: 333 HIV115 Y41 Mutant Construction:

To explore the amino acid sequence necessary for the loss of immunosuppression, HIV115 Y41 mutants were produced by PCR using the pDFG HIV115 WT plasmid as template and pairs of primers 1-42 and 43-2, 1-44 and 45-2, 1-46 and 47-2, 1-48 and 49-2, 1-50 and 51-2, or 1-52 and 53-2, to introduce the Y41K, Y41H, Y41A, Y41G, Y41F, or Y41L mutations respectively. The PCR products were then used as templates in subsequent PCR using primers 1-2. These PCR fragments were digested with SfiI and MluI and inserted into pDFG-ectoSyncytin-1 (Mangeney et al, 2007) opened with the same enzymes

All the constructions were sequenced before use.

N^(o) Name Primer sequence (5′-3′) SEQ ID NO 42 TMHIV115 Y41K as GTTGATCCTTTAGtTtTCTTTCCACAGCCAG SEQ ID NO: 334 43 TMHIV115 Y41K s CTGGCTGTGGAAAGAaAaCTAAAGGATCAAC SEQ ID NO: 335 44 TMHIV115 Y41H as GTTGATCCTTTAGGTgTCTTTCCACAGCCAG SEQ ID NO: 336 45 TMHIV115 Y41H s CTGGCTGTGGAAAGAcACCTAAAGGATCAAC SEQ ID NO: 337 46 TMHIV115 Y41A as GTTGATCCTTTAGGgcTCTTTCCACAGCCAG SEQ ID NO: 338 47 TMHIV115 Y41A s CTGGCTGTGGAAAGAgcCCTAAAGGATCAAC SEQ ID NO: 339 48 TMHIV115 Y41G as GTTGATCCTTTAGGccTCTTTCCACAGCCAG SEQ ID NO: 340 49 TMHIV115 Y41G s CTGGCTGTGGAAAGAggCCTAAAGGATCAAC SEQ ID NO: 341 50 TMHIV115 Y41F as GTTGATCCTTTAGGaATCTTTCCACAGCCAG SEQ ID NO: 342 51 TMHIV115 Y41F s CTGGCTGTGGAAAGATtCCTAAAGGATCAAC SEQ ID NO: 343 52 TMHIV115 Y41L as GTTGATCCTTTAGGagTCTTTCCACAGCCAG SEQ ID NO: 344 53 TMHIV115 Y41L s CTGGCTGTGGAAAGActCCTAAAGGATCAAC SEQ ID NO: 345 HIV115 Y41 Double Mutants Construction:

HIV115 Y41R K72A, G or S double mutants were produced by PCR using the pDFG HIV115 Y41R plasmid as template and pairs of primers 1-94 and 93-2, 1-96 and 95-2, or 1-98 and 97-2. The PCR products were then used as templates in subsequent PCR using primers 1-2. These PCR fragments were digested with SfiI and MluI and inserted into pDFG-ectoSyncytin-1 (Mangeney et al, 2007) opened with the same enzymes. HIV115 R40X Y41R double-mutants were produced by PCR using the pDFG HIV115 Y41R as template and pairs of primers 1-54 and 55-2, 1-56 and 57-2 to introduce the R40A and R40E mutations respectively. HIV115 R40X Y41A double-mutants were produced by PCR using the pDFG HIV115 Y41A as template and pairs of primers 1-58 and 59-2, 1-60 and 61-2, 1-62 and 63-2, 1-64 and 65-2 to introduce the R40A, R40E, R40S, R40T mutations respectively. HIV115 R40X simple mutants were also generated using pDFG HIV115 WT as template and pairs of primers 1-66 and 67-2, 1-68 and 69-2, 1-70 and 71-2, 1-72 and 73-2.

The PCR products were then used as templates in subsequent PCR using primers 1-2. These PCR fragments were digested with SfiI and MluI and inserted into pDFG-ectoSyncytin-1 (Mangeney et al, 2007) opened with the same enzyme.

N^(o) Name Primer sequence (5′-3′) SEQ ID 54 TM CTGGCTGTGGAAgcAcgCCTAAAGGATCAAC SEQ ID HIV115R40AY41R- NO: 378 S 55 TM GTTGATCCTTTAGGcgTgcTTCCACAGCCAG SEQ ID HIV115R40AY41R- NO: 379 AS 56 TM CTGGCTGTGGAAgaAcgCCTAAAGGATCAAC SEQ ID HIV115R40EY41R- NO: 380 S 57 TM GTTGATCCTTTAGGcgTtcTTCCACAGCCAG SEQ ID HIV115R40EY41R- NO: 381 AS 58 TM CTGGCTGTGGAAgctgcCCTAAAGGATCAAC SEQ ID HIV115R40AY41A- NO: 382 S 59 TM GTTGATCCTTTAGGgcagcTTCCACAGCCAG SEQ ID HIV115R40AY41A- NO: 383 AS 60 TM CTGGCTGTGGAAgaagcCCTAAAGGATCAAC SEQ ID HIV115R40EY41A- NO: 384 S 61 TM GTTGATCCTTTAGGgcttcTTCCACAGCCAG SEQ ID HIV115R40EY41A- NO: 385 AS 62 TM CTGGCTGTGGAAagcgcCCTAAAGGATCAAC SEQ ID HIV115R40SY41A- NO: 386 S 63 TM GTTGATCCTTTAGGgcgctTTCCACAGCCAG SEQ ID HIV115R40SY41A- NO: 387 AS 64 TM CTGGCTGTGGAAacagcCCTAAAGGATCAAC SEQ ID HIV115R40TY41A- NO: 388 S 65 TM GTTGATCCTTTAGGgctgtTTCCACAGCCAG SEQ ID HIV115R40TY41A- NO: 389 AS 66 TM CTGGCTGTGGAAgctTACCTAAAGGATCAAC SEQ ID HIV115R40A-S NO: 390 67 TM GTTGATCCTTTAGGTAagcTTCCACAGCCAG SEQ ID HIV115R40A-AS NO: 391 68 TM CTGGCTGTGGAAgaaTACCTAAAGGATCAAC SEQ ID HIV115R40E-S NO: 392 69 TM GTTGATCCTTTAGGTAttcTTCCACAGCCAG SEQ ID HIV115R40E-AS NO: 393 70 TM CTGGCTGTGGAAagcTACCTAAAGGATCAAC SEQ ID HIV115R40S-S NO: 394 71 TM GTTGATCCTTTAGGTAgctTTCCACAGCCAG SEQ ID HIV115R40S-AS NO: 395 72 TM CTGGCTGTGGAAacaTACCTAAAGGATCAAC SEQ ID HIV115R40T-S NO: 396 73 TM GTTGATCCTTTAGGTAtgtTTCCACAGCCAG SEQ ID HIV115R40T-AS NO: 397 93 TMHIV115K72A GCTAGTTGGAGTAATgcATCTCTGGAACAGATTTGG SEQ ID s NO: 398 94 TMHIV115K72A CCAAATCTGTTCCAGAGATgcATTACTCCAACTAGC SEQ ID as NO: 399 95 TMHIV115K72G GCTAGTTGGAGTAATggATCTCTGGAACAGATTTGG SEQ ID s NO: 400 96 TMHIV115K72G CCAAATCTGTTCCAGAGATccATTACTCCAACTAGC SEQ ID as NO: 401 97 TMHIV115K72S GCTAGTTGGAGTAATtcATCTCTGGAACAGATTTGG SEQ ID s NO: 402 98 TMHIV115K72S CCAAATCTGTTCCAGAGATgaATTACTCCAACTAGC SEQ ID as NO: 403 Introduction of HIV115 Mutations into an HIV Env Expression Vector:

To introduce these HIV envelope mutations into the HIVenv pTr712 expression vector (Schnierle et al PNAS 1997), a silent mutation was first generated to create an SfiI insertion site. PCR fragments were generated using pTr712as a template and primer pairs 38-39 and 40-41. The PCR products were then used as templates in subsequent PCR using primers 38-41.

The resulting PCR fragment was then digested with BsaBI and HindIII and inserted into the pTr712 plasmid opened with the same enzymes resulting in the pTr712-Sfi plasmid. PCR fragments of each HIV115 mutation were then generated by using primers 40-41 and the pDFG HIV115 mutant plasmids as templates, digestion with SfiI and HindIII and then insertion into the pTr712-Sfi plasmid opened with the same enzymes.

All the constructions were sequenced before use.

N^(o) Name Primer sequence (5′-3′) SEQ ID NO 38 HIV BH10 BsaBI s acaaattagatgttcatcaaatattacaggg SEQ ID NO: 346 39 HIV BH10 SfiI mutsil as GCAACAGATGCTGTTGgGCCTCA SEQ ID NO: 347 ATgGCCCTCAGCAAATTGTTC 40 HIV BH10 SfiI mutsil s GAACAATTTGCTGAGGGCcATTG SEQ ID NO: 348 AGGCcCAACAGCATCTGTTGC 41 HIV BH10 HindIII as gtgtattaagcttgtgtaattgttaatttctc SEQ ID NO: 349 74 EnvHIVTr712-Xho- NNNNNCTCGAGACCGGTccaactaga SEQ ID NO: 404 Age-Kozak-S accATGAGAGTGAAGGAGAAATA TCAGC 75 EnvHIVTr712-Mlu-AS NNNNNACGCGTTCAATATCCCTG SEQ ID NO: 405 CCTAACTC 76 EnvHIVWT-Mlu-AS NNNNNACGCGTTTATAGCAAAAT SEQ ID NO: 406 CCTTTCCAAGC Establishment of Env-Expressing Tumor Cells and MCA205 Tumor-Rejection Assay:

7.5×10⁵ 293 T cells were cotransfected with the env-expressing pDFG retroviral vector to be tested (1.75 μg) and expression vectors for the MLV proteins (0.55 μg for the amphotropic MLV env vector and 1.75 μg for the MLV gag and pol vector). 36 hours post-transfection, supernatants were harvested for infection of MCA205 tumor cells (2.5 ml of supernatant per 5×10⁵ cells with 8 μg/ml polybrene). Cells were maintained in selective medium (400 units/ml hygromycin) for 3 weeks, and then washed with PBS, trypsinized and inoculated subcutaneously in the shaved area of each mouse right flank as in Mangeney et al (1998, 2007). Tumor growth was monitored by palpation twice or thrice weekly and tumor area (mm²) determined by measuring perpendicular tumor diameters. The extent of “immunosuppression” was quantified by an index based on tumor size: (A_(env)−A_(none))/A_(none), where A_(env) and A_(none) are the mean areas at the peak of growth of tumors from Balb/c mice injected with env-expressing or control cells, respectively.

Analysis of HIVenv Expression:

293T cells were transfected with 4 mg of the expression vector for the HIV-1 envelope (pTr712) either wild-type or mutated at the indicated positions, by Calcium Phosphate precipitation. Cells are washed 16 h later and then harvested 2 days post-transfection using PBS-EDTA 5 mM. The 110-H monoclonal antibody (anti-V3 loop, gift from Hybridolab, Pasteur Institute) was used ( 1/200 dilution) to stain the HIV envelope. As a secondary antibody, the Inventors used the goat anti mouse IgG Alexa 488 ( 1/400) (Invitrogen). For intracellular HIVenv staining, 293T cells were fixed with a formaldehyde buffer and then permeabilized (BD cyto fix/cytoperm, BD Biosciences). The isotype mouse anti IgG1Kappa (BD Biosciences) was used to control non-specific staining Fluorescence was acquired by flow cytometry using a FACS Calibur (BD Biosciences), and data analysed by the CellQuest software (BD Biosciences).

Mutated Env Pseudotyping and Measure of Viral Titer:

293T cells are triple transfected with 3 mg of a reporter MLV vector carrying GFP (CNCG), 1.75 mg Mo-MLV gag-pol vector and 0.55 mg phCMV vector encoding HIV-1 envelope wt or mutated at the indicated positions. The infectivity of Mo-MLV virions pseudotyped with HIV-1 Env, harvested 48 hours post-transfection, is measured using U87 cells (CD4⁺, CXCR4⁺) as target cells. The infectivity of the envelopes is analysed after 72 h exposure diluted 0.45 mm-filtered supernatant in presence of 4 mg/mL polybrene. The fluorescence (GFP) is acquired by flow cytometry using a FACS Calibur (BD Biosciences). The results are analysed by the CellQuest software (BD Biosciences). The resulting titers (number of infected cells/mL) are calculated as the following: (% GFP⁺ cells (infected) x plated cell number)/volume of supernatant×1000.

Env SIV Mutants Construction:

PCR fragments were generated using p239 SPE3′ (the plasmid encoding the SIV half virus containing the envelope protein) as a template and primer pairs 77-79 and 78-80, 77-81 and 78-82, 77-83 and 78-84, 77-85 and 78-86, 77-87 and 78-88, 77-89 and 78-90 to introduce the E39R, K40R, Y41R, L42R, K43R, D44R mutations respectively. The PCR products were the used as templates in subsequent PCR using primers 77-78. The resulting PCR fragment was then digested with BmgBI and NheI and inserted into the p239 SPE3′ plasmid opened with the same enzymes. All the constructions were sequenced before use.

Introduction of SIV Mutants Ectodomain into pDFG:

To generate the pDFG plasmids encoding the fragment of SIV envelope ectodomain-55, PCR fragments generated using p239 SPE3′ WT and mutants as a template and primer pairs 91-92, were digested with SfiI and MluI and inserted into pDFG opened with the same enzymes.

77 Env SIV S SEQ ID NO: 350 ccgctcagtcccgaactttattggc 78 Env SIV AS SEQ ID NO: 351 ggtggggaagagaacactggcc 79 SIV env E39R s SEQ ID NO: 352 cagactagggtcactgccatcCGCaagtacttaaaggaccaggcg 80 SIV env E39R as SEQ ID NO: 353 cgcctggtcctttaagtacttGCGgatggcagtgaccctagtctg 81 SIV env K40R s SEQ ID NO: 354 actagggtcactgccatcgagCGCtacttaaaggaccaggcgcag 82 SIV env K40R as SEQ ID NO: 355 ctgcgcctggtcctttaagtaGCGctcgatggcagtgaccctagt 83 SIV env Y41R s SEQ ID NO: 356 GCCATCGAGAAGcgCTTAAAGGACCAGGCG 84 SIV env Y41R as SEQ ID NO: 357 CGCCTGGTCCTTTAAGcgCTTCTCGATGGC 85 SIV env L42R s SEQ ID NO: 358 gtcactgccatcgagaagtacCGCaaggaccaggcgcagctg 86 SIV env L42R as SEQ ID NO: 359 cagctgcgcctggtccttGCGgtacttctcgatggcagtgac 87 SIV env K43R s SEQ ID NO: 360 gccatcgagaagtacttaCGCgaccaggcgcagctgaatgcttgg  88 SIV env K43R as SEQ ID NO: 361 ccaagcattcagctgcgcctggtcGCGtaagtacttctcgatggc 89 SIV env D44R s SEQ ID NO: 362 gagaagtacttaaagCGCcaggcgcagctgaatgcttgg 90 SIV env D44R as SEQ ID NO: 363 attcagctgcgcctgGCGctttaagtacttctcgatggc 91 TMSIV55 Sfi S SEQ ID NO: 364 ACATggcccagccggccgctgggatagtgcagcaac 92 TMSIV55 Mlu AS SEQ ID NO: 365 GTATacgcgtTTAaaacgcacatccccaagcattc

Comparative Example: Test of the In Vivo Effect of the Mutation G49R, which Corresponds to the Mutation “G19R” in the International Application WO 2010/022,740

WO 2010/022,740 discloses a consensus sequence of 50 amino acid of the HIV ENV protein. In this sequence, it is suggested that substitution of amino acids in positions 10, 19, 24, 34 and 40 affect the immunosuppressive properties of the HIV ENV protein.

These mutations are a transposition in lentivirus of the teaching of WO 2005/095,442 limited to endogenous or onco retroviruses. The authors of WO 2005/095,442 are also the authors of the present application and they early observed that such a transposition is not effective.

Despite the fact that any amino acids can be assigned to the positions 10, 19, 24, 34 or 40 in the consensus sequence of WO 2010/022,740, only one substitution (one residue for one position) was tested by ex vivo experiments in WO 2010/022,740. It is the mutation “G19R”, which corresponds to the mutation G49R in the present FIGS. 1 and 3.

Because the immune response of an individual involves different organs and different cellular and non-cellular components, ex vivo results have no predictive value concerning the in vivo immunosuppressive properties of a viral protein.

To determine if the mutations previously disclosed in WO 2010/022,740 are suitable for a medical use, the mutation G49R has been tested using the MCA205 tumor rejection in vivo assay, as defined in the section “Establishment of env-expressing tumor cells and MCA205 tumor-rejection assay” of the Example.

As shown in FIG. 3, the peptide G49R remains almost as immunosuppressive in vivo as the wild type HIV 115 peptide, with an immunosuppression index which is higher than 0.5. Thus, the mutation G49R does not induce a significant decrease of the in vivo immunosuppressive properties of the HIV ENV protein, with a ratio of immunosuppression index of G49R mutated ENV HIV 115 peptide (i_(mutated env))/immunosuppression index of wild type ENV HIV 115 peptide (i_(wild type env)), which is 0.7 (i.e. higher than 0.5).

This result demonstrates the insufficiently described teaching of WO 2010/022740 since the only one mutation tested in WO 2010/022,740, using an ex vivo test, does not significantly affect the in vivo immunosuppressive properties of the HIV ENV protein.

As a consequence, WO 2010/022,740 raises the same technical problem as the present invention but does not offer a technical solution. 

The invention claimed is:
 1. A pharmaceutical composition comprising as an active substance: a) an isolated mutated human or simian lentiviral ENV protein having no or a reduced immunosuppressive activity as compared to a corresponding wild type non-mutated human or simian lentiviral ENV protein, said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of the corresponding wild type non-mutated human or simian lentiviral ENV protein, said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence: (SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

wherein, X represents any amino acid, and either X_(a) is A, F, G, L or R, and X_(b) is any amino acid, X_(a) is any amino acid, and X_(b) is A, F, G or R, or X_(a) is A, F, G, L or R, and X_(b) is A, F, G or R, or b) at least one fragment of said isolated mutated human or simian lentiviral ENV protein having no or a reduced immunosuppressive activity as compared to the corresponding wild type non-mutated human or simian lentiviral ENV protein, said fragment comprising at least 40 amino acids, said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence: (SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

wherein, X represents any amino acid, and either X_(a) is A, F, G, L or R, and X_(b) is any amino acid, X_(a) is any amino acid, and X_(b) is A, F, G or R, or X_(a) is A, F, G, L or R, and X_(b) is A, F, G or R, in association with a pharmaceutically acceptable carrier, said no or reduction of immunosuppressive activity of the above mentioned mutated human or simian lentiviral ENV protein or of the above defined fragment being liable to be assessed by the fact that in an in vivo assay involving engrafted tumor cells rejection, said tumor cells being transduced either so as to express said mutated ENV protein or said fragment (mutated ENV tumor cells), or said tumor cells being transduced so as to express the corresponding wild type non-mutated ENV protein or a fragment thereof (wild type ENV tumor cells), or said tumor cells being not transduced (normal tumor cells), the following ratio: immunosuppression index of said mutated ENV protein or of said fragment (i_(mutated env))/immunosuppression index of wild type ENV protein (i_(wild type env)) is less than 0.5, i_(mutated env) being defined by: (maximum area reached by mutated ENV tumor cells−maximum area reached by normal tumor cells)/(maximum area reached by normal tumor cells), and i_(wild type env) being defined by: (maximum area reached by wild type ENV tumor cells−maximum area reached by normal tumor cells)/(maximum area reached by normal tumor cells).
 2. The pharmaceutical composition according to claim 1, comprising as an active substance: a) an isolated mutated human or simian lentiviral ENV protein having no or a reduced immunosuppressive activity as compared to a corresponding wild type non-mutated human or simian lentiviral ENV protein, said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of the corresponding wild type non-mutated human or simian lentiviral ENV protein, said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence: (SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

wherein X represents any amino acid, and either X_(a) is A, F, G, L or R, and X_(b) is L, I, V, M or P, or X_(a) is Y, I, H, C or T, and X_(b) is A, F, G or R, or X_(a) is A, F, G, L or R, and X_(b) is A, F, G or R, or b) at least one fragment of said isolated mutated human or simian lentiviral ENV protein having no or a reduced immunosuppressive activity as compared to a corresponding wild type non-mutated human or simian lentiviral ENV protein, said fragment comprising at least 40 amino acids, said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence: (SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

wherein X represents any amino acid, and either X_(a) is A, F, G, L or R, and X_(b) is L, I, V, M or P, or X_(a) is Y, I, H, C or T, and X_(b) is A, F, G or R, or X_(a) is A, F, G, L or R, and X_(b) is A, F, G or R, in association with a pharmaceutically acceptable carrier.
 3. The pharmaceutical composition according to claim 1, comprising as an active substance: a) an isolated mutated human or simian lentiviral ENV protein having no or a reduced immunosuppressive activity as compared to a corresponding wild type non-mutated human or simian lentiviral ENV protein, said mutated human or simian lentiviral ENV protein resulting from mutation of the transmembrane (TM) subunit of the corresponding wild type non-mutated human or simian lentiviral ENV protein, said mutated human or simian lentiviral ENV protein comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence: (SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

wherein X represents any amino acid, and either X_(a) is A, F, G, L or R, and X_(b) is L, or X_(a) is Y, and X_(b) is A, F, G or R, or b) at least one fragment of said isolated mutated human or simian lentiviral ENV protein having no or a reduced immunosuppressive activity as compared to a corresponding wild type non-mutated human or simian lentiviral ENV protein, said fragment comprising at least 40 amino acids, said fragment comprising a mutated immunosuppressive domain (ISU) containing the following amino acid sequence: (SEQ ID NO: 416) A-[I/V]-E-[K/R]-X_(a)-X_(b)-X-D-Q,

wherein X represents any amino acid, and either X_(a) is A, F, G, L or R, and X_(b) is L, or X_(a) is Y, and X_(b) is A, F, G or R, in association with a pharmaceutically acceptable carrier.
 4. The pharmaceutical composition according to claim 1, wherein said isolated mutated human or simian lentiviral ENV protein or said fragment of said isolated mutated human or simian lentiviral ENV protein comprises one of the following amino acid sequences: (SEQ ID NO: 422) A-I-E-K-X_(a)-X_(b)-X-DQ, (SEQ ID NO: 423) A-I-E-R-X_(a)-X_(b)-X-DQ, (SEQ ID NO: 424) A-V-E-K-X_(a)-X_(b)-X-DQ, (SEQ ID NO: 425) A-V-E-R-X_(a)-X_(b)-X-DQ.


5. The pharmaceutical composition according to claim 1, wherein said isolated mutated human or simian lentiviral ENV protein, or said fragment of said isolated mutated human or simian lentiviral ENV protein, comprises one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO: 42, SEQ ID NO: 71, SEQ ID NO: 9 to 12, SEQ ID NO: 14 to 41, SEQ ID NO: 43 to 70, and SEQ ID NO: 72 to
 269. 6. The pharmaceutical composition according to claim 1, in a formulation for its use for inhibiting lentiviral infection.
 7. The pharmaceutical composition according to claim 1, wherein said isolated mutated human or simian lentiviral ENV protein, or said fragment of said isolated mutated human or simian lentiviral ENV protein, comprises one of the amino acid sequences selected from the group consisting of SEQ ID NO: 96-211.
 8. The pharmaceutical composition according to claim 1, wherein said isolated mutated human or simian lentiviral ENV protein, or said fragment of said isolated mutated human or simian lentiviral ENV protein, comprises one of the amino acid sequences selected from the group consisting of SEQ ID NO: 212-269.
 9. The pharmaceutical composition according to claim 6, wherein the lentiviral infection is HIV-1 infection, HIV-2 infection or SIV infection.
 10. The pharmaceutical composition according to claim 6, wherein the formulation is capable of inducing an anti-lentiviral immune response against HIV-1 infection, HIV-2 infection or SIV infection. 